Graduate Theses and Dissertations
Graduate College
2011
Mouth rinsing with branched-chain amino acids
and carbohydrates on steady-state metabolism,
exercise performance, and the endocrine responses
to exercise
Zebblin Matthew Sullivan
Iowa State University
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Sullivan, Zebblin Matthew, "Mouth rinsing with branched-chain amino acids and carbohydrates on steady-state metabolism, exercise
performance, and the endocrine responses to exercise" (2011). Graduate Theses and Dissertations. Paper 10210.
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Mouth rinsing with branched-chain amino acids and carbohydrates on steady-state
metabolism, exercise performance, and the endocrine responses to exercise
by
Zebblin Matthew Sullivan
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Kinesiology (Biological Basis of Physical Activity)
Program of Study Committee:
Douglas S. King, Major Professor
Rick L. Sharp
Amy S. Welch
Warren D. Franke
Ruth E. Litchfield
Iowa State University
Ames, Iowa
2011
Copyright © Zebblin Matthew Sullivan, 2011. All rights reserved
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TABLE OF CONTENTS
LIST OF FIGURES
iv
LIST OF TABLES
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CHAPTER 1. GENERAL INTRODUCTION
Introduction
Dissertation Organization
References
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CHAPTER 2. LITERATURE REVIEW
Introduction
BCAA Catabolism
Central Fatigue Theory
Carbohydrates, BCAA, and Central Fatigue
BCAA and Anaerobic Performance
BCAA and Buffer Capacity
Responders to BCAA Supplementation
Need for Additional BCAA Intake
BCAA and Cognition
BCAA and Physical Activity
Leucine Supplementation and Obesity
BCAA and Muscle Recovery
Safety of BCAA Consumption
BCAA Summary
Carbohydrate Mouth Rinse
Cephalic Phase of Insulin Release
Mouth Rinse Summary
References
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CHAPTER 3. MOUTH RINSING WITH BRANCHED-CHAIN
AMINO ACIDS AND CARBOHYDRATES ON STEADY-STATE
METABOLISM, EXERCISE PERFORMANCE, AND THE ENDOCRINE
RESPONSES TO EXERCISE
Abstract
Introduction
Methods
Results
Discussion
Practical Applications
References
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CHAPTER 4. GENERAL CONCLUSIONS
Discussion and Recommendations for Future Research
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APPENDIX. BRANCHED-CHAIN AMINO ACID SUPPLEMENTATION
ATTENUATES THE LOSS OF MUSCLE POWER AFTER ECCENTRIC
EXERCISE IN MIDDLE-AGED WOMEN
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ACKNOWLEDGMENTS
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LIST OF FIGURES
Figure 1A. Evaluation of BCAA, CHO, and PLA bitterness pre- and post-steady-state
exercise
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Figure 1B. Evaluation of BCAA, CHO, and PLA bitterness pre- and post-cycle time trial 95
Figure 2A. Evaluation of BCAA, CHO, and PLA sweetness pre- and post-steady-state
exercise
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Figure 2B. Evaluation of BCAA, CHO, and PLA sweetness pre- and post-cycle time trial 96
Figure 3A. CHO oxidation rates in response to mouth rinsing with BCAA, CHO, and PLA
during steady-state exercise
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Figure 3B. CHO oxidation rates in response to mouth rinsing with BCAA, CHO, and PLA
during the cycle time trial
97
Figure 4A. Fat oxidation rates in response to mouth rinsing with BCAA, CHO, and PLA
during steady-state exercise
98
Figure 4B. Fat oxidation rates in response to mouth rinsing with BCAA, CHO, and PLA
during the cycle time trial
98
Figure 5A. Plasma insulin response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise
99
Figure 5B. Plasma insulin response to mouth rinsing with BCAA, CHO, and PLA during the
cycle time trial
99
Figure 6A. Plasma prolactin response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise
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Figure 6B. Plasma prolactin response to mouth rinsing with BCAA, CHO, and PLA during
the cycle time trial
100
Figure 7A. Plasma lactate response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise
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Figure 7B. Plasma lactate response to mouth rinsing with BCAA, CHO, and PLA during the
cycle time trial
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Figure 8A. Plasma glucose response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise
102
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Figure 8B. Plasma glucose response to mouth rinsing with BCAA, CHO, and PLA during the
cycle time trial
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Figure 9. Time to complete the cycle time trial when mouth rinsing with BCAA, CHO, and
PLA
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LIST OF TABLES
Table 1. Analysis of three d diet records
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Table 2. Heart rate (HR), ratings of perceived exertion (RPE), feeling scale (FS), and felt
arousal activation (FAS) during steady-state exercise
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Table 3. Effects of mouth rinsing with BCAA and CHO on heart rate (HR), ratings of
perceived exertion (RPE), feeling scale (FS), and felt arousal activation (FAS) during
cycle time trial
94
1
CHAPTER 1. GENERAL INTRODUCTION
Introduction
A vast amount of literature exists on the effects of purported ergogenic aids
(performance enhancing drugs and supplements). Historic examples of athletes attempting to
maximize performance include Roman and Greek athletes being advised by physicians to eat
raw meat to maintain their “animal competitiveness,” and marathon runners in the 1908
Olympics allegedly consuming brandy to improve performance (McArdle, Katch, & Katch,
2010). In a survey of 53 coaches and athletic trainers at a Division I university, 94% of
athletes were provided nutritional supplements (Smith-Rockwell et al., 2001).
Consumption of carbohydrates may delay the onset of fatigue and improve aerobic
exercise performance (Coggan & Coyle, 1987; Coyle et al., 1986). Carbohydrate ingestion
may be especially beneficial during prolonged exercise (> 2 h) due to maintenance of the
carbohydrate oxidation rate (Coyle et al., 1986) and prevention of hypoglycemia (Ivy et al.,
1979). Carbohydrate ingestion may also improve performance in shorter, high-intensity
exercise (< 1 h; Neufer et al., 1987; Jeukendrup, 2004). Whereas the underlying mechanism
for the improvements in exercise performance during shorter exercise bouts in which
glycogen depletion is unlikely has not been elucidated, there may be central or nonmetabolic
factors involved.
Previous research has demonstrated that mouth rinsing with a carbohydrate solution
enhances performance (Carter et al., 2004; Pottier et al., 2008; Rollo et al., 2008; Chambers
et al., 2009; Rollo et al., 2010). Although the mechanisms for the improvement in
performance are not yet defined, it has been hypothesized that presentation of energy to the
mouth may stimulate reward centers of the brain that increase motivational drive and
2
exercise performance. Chambers et al. (2009) showed that non-sweet carbohydrate
(maltodextrin) and glucose in the mouth produced similar cortical activation at rest and both
solutions improved exercise performance, suggesting that mouth rinsing with both sweet and
non-sweet solutions may improve performance by activating brain centers responsible for
motor output and drive. Unidentified oral receptors may exist that respond to the caloric
property of carbohydrate, independent of carbohydrate sweetness properties.
The cephalic phase of insulin release (CPIR) has been observed in animals and
humans. The β-cells of the pancreas secrete insulin before an increase in plasma glucose.
Oral cavity stimulation with sweet, non-caloric substances stimulates the CPIR without
affecting plasma glucose concentrations. Oral cavity stimulation with non-sweet, caloriccontaining substances does not stimulate the CPIR or affect plasma glucose concentrations
(Tonosaki et al., 2007). It has been hypothesized that mouth rinsing with sweet carbohydrates
may stimulate insulin secretion during exercise, potentially improving exercise performance.
Branched-chain amino acids (BCAA; leucine, isoleucine, and valine)
supplementation may be beneficial. Blomstrand (2001) suggested that the benefits of BCAA
use are likely to be greater during activities in which the central component of fatigue plays a
considerable role, such as exercise in the heat or during a competitive race. Plasma
tryptophan is the amino acid precursor in the synthesis of 5-hydroxytryptamine (5-HT;
serotonin) in the brain, and is involved in the control of arousal, sleepiness, and mood
(Young, 1986). Increased free-tryptophan concentrations lead to increased serotonin
synthesis, which is associated with lethargy, fatigue, and decreased exercise performance
(Davis et al., 2000). Branched-chain amino acids may limit central fatigue by competing with
free-tryptophan for uptake into the brain via the same transport mechanism (Pardridge and
3
Oldendorf, 1975; Blomstrand et al., 1997). Branched-chain amino acids supplementation has
been shown to decrease perceived exertion and increase endurance performance (Blomstrand
et al., 1991, 1997) and reduce brain serotonin synthesis (Gomez-Merino et al., 2001; Smriga
et al. 2006). Whereas research has shown that BCAA supplementation may attenuate
exercise-induced fatigue, some studies fail to corroborate these findings (Wagenmakers,
1992; Varnier et al., 1994; Strüder et al., 1998).
This dissertation research will determine if mouth rinsing with BCAA enhances
exercise performance. This research will assess the extent to which improvement in
performance is due to carbohydrates per se, or the presentation of energy to the oral cavity.
Additionally, it is not currently known what effects rinsing the mouth with carbohydrates and
branched-chain amino acids have on the concentration of blood glucose and lactate during
exercise, the steady-state metabolic response to exercise, and the pancreatic secretion of
insulin during exercise. Another major aim of this dissertation research is to determine the
effects of mouth rinsing with carbohydrate and BCAA during exercise on markers of central
(neural-based) fatigue. Plasma prolactin has been shown to be a measure of central serotonin
levels (Yatham and Steiner, 1993) and will be used as the blood marker of central fatigue in
the study. Finally, the effects of rinsing the mouth with carbohydrates and branched-chain
amino acids on the subjective exercise experience and performance responses to exercise will
be examined in the dissertation research.
Dissertation Organization
This dissertation is organized into a General Introduction, Review of Literature,
Manuscript, General Conclusions, and an Appendix containing an additional manuscript
prepared for journal submission. The primary author for the manuscript is Zebblin M.
4
Sullivan, a Ph.D. student in the Kinesiology department at Iowa State University. Dr.
Douglas S. King, Professor of Kinesiology at Iowa State University contributed to the
experimental design, data analysis, and preparation of the manuscript.
References
Blomstrand E, Hassmén P, Ek S, Ekblom B, Newsholme EA. Influence of ingesting a
solution of branched-chain amino acids on perceived exertion during exercise. Acta Physiol
Scand 159(1): 41-49, 1997.
Blomstrand E, Hassmén P, Ekblom B, Newsholme EA. Administration of branched-chain
amino acids during sustained exercise – effects on performance and on plasma concentration
of some amino acids. Eur J Appl Physiol Occup Physiol 63(2): 83-88 1991.
Blomstrand E. Amino acids and central fatigue. Amino acids 20: 25-34, 2001.
Carter JM, Jeukendrup AE, Jones DA. The effect of carbohydrate mouth rinse on 1-h
cycle time trial performance. Med Sci Sports Exerc 36(12): 2107-2111, 2004.
Chambers ES, Bridge MW, Jones DA. Carbohydrate sensing in the human mouth: effects
on exercise performance and brain activity. J Physiol 587(Pt 8): 1779-1794, 2009.
Coggan AR, Coyle EF. Reversal of fatigue during prolonged exercise by carbohydrate
infusion or ingestion. J Appl Physiol 63(6): 2388-2395, 1987.
Cole KJ, Costill DL, Starling RD, Goodpaster BH, Trappe SW, Fink WJ. Effect of
caffeine ingestion on perception of effort and subsequent work production. Int J Sport Nutr
6(1): 14-23, 1996.
Davis JM, Alderson NL, Welsh RS. Serotonin and central nervous system fatigue:
nutritional considerations. Am J Clin Nutr 72(2 Suppl): 573S-578S, 2000.
Gomez-Merino D, Béquet F, Berthelot M, Riverain S, Chennaoui M, Guezennec CY.
Evidence that the branched-chain amino acid L-valine prevents exercise-induced release of
5-HT in rat hippocampus. Int J Sports Med 22(5): 317-322, 2001.
Jeukendrup AE. Carbohydrate ingestion during exercise and performance. Nutrition, 20(7):
669-677, 2004.
McArdle WD, Katch FI, Katch VL. Exercise Physiology: Nutrition, Energy, and Human
Performance, 7th ed., Philadelphia: Lippincott Williams & Wilkins, 2010.
5
Neufer PD, Costill DL, Flynn MG, Kirwan JP, Mitchell JB, Houmard J. Improvements
in exercise performance: effects of carbohydrate feedings and diet. J Appl Physiol 62(3):
983-988, 1987.
Pardridge WM, Oldendorf WH. Kinetic analysis of blood-brain barrier transport of amino
acids. Biochim Biophys Acta 401(1): 128-136, 1975.
Pottier A, Bouckaert J, Gilis W, Roels T, Derave W. Mouth rinse but not ingestion of a
carbohydrate solution improves 1-h cycle time trial performance. Scand J Med Sci Sports
20(1): 105-111, 2008.
Rollo I, Cole M, Miller R, Williams C. Influence of mouth rinsing a carbohydrate solution
on 1-h running performance. Med Sci Sports Exerc 42(4): 798-804, 2010.
Rollo I, Williams C, Gant N, Nute M. The influence of carbohydrate mouth rinse on selfselected speeds during a 30-min treadmill run. Int J Sport Nutr Exerc Metab 18(6): 585-600,
2008.
Smith-Rockwell M, Nickols-Richardson SM, Thye FW. Nutrition, knowledge, opinions,
and practices of coaches and athletic trainers at a division 1 university. Int J Sport Nutr Exerc
Metab 11(2): 174-185, 2001.
Smriga M, Kameishi M, Torii K. Exercise-dependent preference for a mixture of branchedchain amino acids and homeostatic control of brain serotonin in exercising rats. J Nutr
136(2): 548S-552S, 2006.
Strüder HK, Hollmann W, Platen P, Donike M, Gotzmann A, Weber K. Influence of
paroxetine, branched-chain amino acids and tyrosine on neuroendocrine system responses
and fatigue in humans. Horm Metab Res 30(4): 188-194, 1998.
Tonosaki K, Hori Y, Shimizu Y, Tonosaki, K. Relationships between insulin release and
taste. Biomed Res 28(2): 79-83, 2007.
Varnier M, Sarto P, Martines D, Lora L, Carmignoto F, Leese GP, Naccarato R. Effect
of infusing branched-chain amino acid during incremental exercise with reduced muscle
glycogen content. Eur J Appl Physiol Occup Physiol 69(1): 26-31, 1994.
Wagenmakers AJM, Brookes JH, Coakley JH, Reilly T, Edwards RHT. Exerciseinduced activation of branched-chain 2-oxo acid dehydrogenase in human muscle. Eur J
Appl Physiol 59(3): 159-167, 1989.
Varnier M, Sarto P, Martines D, Lora L, Carmignoto F, Leese GP, Naccarato R. Effect
of infusing branched-chain amino acid during incremental exercise with reduced muscle
glycogen content. Eur J Appl Physiol Occup Physiol 69(1): 26-31, 1994.
6
Yatham LN, Steiner M. Neuroendocrine probes of serotonergic function: a critical review.
Life Sci 53(6): 447-463, 1993.
Young SN. 1986. The clinical psychopharmacology of tryptophan. In: Nutrition and the
Brain (vol 7), edited by Wurtman RJ, Wurtman JJ. New York: Raven Press, 1986, p. 49-88.
7
CHAPTER 2. LITERATURE REVIEW
Introduction
Nutritional ergogenic aids can be classified into four categories: substances that
promote anabolism and improve body composition, substances that provide quickly utilizable
energy, substances that facilitate recovery from physical exhaustion, and substances that fill
other important roles in the physiology of exercise (Williams, 1995). Leucine, isoleucine, and
valine are named branched-chain amino acids (BCAA) because their respective side chains
are similarly branched in structure, unlike the other protein amino acids that have linear side
chains. The BCAA are three of the nine essential amino acids for humans and must therefore
be consumed in the diet. Branched-chain amino acids account for about 35% of the essential
amino acids in muscle proteins (Harper et al., 1984).
Leucine has been investigated more intensely than isoleucine or valine mainly
because its rate of oxidation is higher than the other two BCAA (Mero, 1999). Whereas the
other essential amino acids are oxidized primarily in the liver, BCAA can be oxidized in
skeletal muscle (Rennie, 1996). During exercise, BCAA contribute to energy metabolism by
providing intermediates for the tri-carboxylic acid (TCA) cycle. Additionally, BCAA may be
transaminated to their respective α-keto acids, which are used for gluconeogenesis during
exercise. The efficacy of BCAA supplementation on exercise performance has been posited
to depend on several factors including the mode of administration (Verger et al., 1994), the
nutritional status of recipients (Calders et al., 1997), use in conjunction with carbohydrates
(Blomstrand, 2001), and the length and intensity of exercise (Strüder et al. 1998). Blomstrand
(2001) argues that the benefits of BCAA use are likely to be greater during activities in
8
which the central component of fatigue plays a considerable role, such as exercise in the heat
or during a competitive race.
Branched-chain amino acids can be obtained from four sources: whole-food proteins,
dietary protein supplements, solutions of protein hydrolysates, and free amino acids
(Gleeson, 2005). There are four methods of consuming high amounts of BCAA – greater
total energy intakes, deliberate consumption of high-protein diets and protein supplements,
consumption of protein hydrolysates or mixtures of essential amino acids before and after
exercise, and consumption of drinks containing BCAA (Gleeson, 2005). High levels of
protein, amino acids, and/or BCAA may be consumed for one of several reasons. People
wishing to increase muscle mass or lean tissue often consume high levels of dietary protein.
Bodybuilders and weightlifters may consume up to 3 g/kg body mass of dietary protein
(Jeukendrup & Gleeson, 2004). Infusion of BCAA, particularly leucine, has been shown to
increase protein synthesis (Alvestrand et al., 1990) and decrease protein breakdown (Louard
et al., 1990; Nair et al., 1992), potentially resulting in net protein accretion. Leucine
stimulates the mammalian turnover of rapamycin (mTOR), a serine-threonine kinase
involved in protein synthesis (Anthony et al., 2001; Kimball & Jefferson, 2006).
Additionally, infusion of leucine into the third ventricle of the rat brain reduced voluntary
food intake and body mass through activation of the mTOR pathway (Cota et al., 2006).
Because prolonged exercise bouts increase BCAA uptake into muscle for oxidation for
energy, endurance athletes may consumer higher amounts of dietary protein to meet the
greater demands of BCAA oxidation. However, whereas there is a two- to three-fold increase
in BCAA oxidation during exercise, the oxidation of carbohydrate and fat increases much
more so, 10- to 20-fold during exercise (Wolfe et al., 1982; Knapik et al., 1991).
9
Furthermore, the activities of enzymes of BCAA oxidation are likely too low to allow a
major contribution to energy expenditure from BCAA (Wagenmakers et al., 1989;
Wagenmakers et al., 1991). Despite high intakes of protein being common among athletes,
there is still much debate on the efficacy of higher protein intake on performance benefits.
Branched-chain amino acids donate nitrogen for glutamine synthesis, helping to maintain
plasma glutamine concentrations during exercise, which may enhance or strengthen the
immune system. Some weak experimental evidence suggests that BCAA supplementation
may attenuate immune system depression caused by prolonged strenuous exercise (Bassit et
al., 2002). However, the strength and validity of the experimental design and statistical
analysis of that study has been questioned (Calder, 2006). Further research is warranted in
the area of BCAA supplementation and immune function.
Catabolism of BCAA
Branched-chain amino acids are essential for protein synthesis; however, some
intermediates (branched-chain α-keto acids; BCKA) in their catabolism can be toxic at high
concentrations (Harper et al., 1984). It is therefore imperative that excess BCAA are disposed
of for maintenance of normal body function. The entire catabolism of BCAA occurs in the
mitochondria. The first step in BCAA catabolism involves the reversible transamination of
BCAA to produce BCKA, catalyzed by the enzyme branched-chain aminotransferase (Figure
1). The next reaction in the catabolism of BCAA is the rate-limiting step of the pathway. This
irreversible reaction, catalyzed by the enzyme branched-chain α-keto acid dehydrogenase
(BCKDH) complex, involves the oxidative decarboxylation of BCKA to coenzyme A
compounds. Because this is the rate-limiting reaction of BCAA catabolism, it is tightly
regulated (Harris et al., 1990). The branched-chain α-keto acid dehydrogenase complex is
10
activated when dephosphorylated by BCKDH phosphatase and inactivated when
phosphorlyated by BCKDH kinase. Apparently, BCKDH kinase is the primary regulator of
the activation status of the BCKDH complex (Shimomura et al., 2001). The transamination
of leucine forms α-ketoisocaproate (KIC), a potent inhibitor of BCKDH kinase (Paxton and
Harris, 1984). Therefore, when KIC accumulates in tissues, the catabolism of branched-chain
amino acids is stimulated.
Branched-chain amino acids
(Valine, Isoleucine, Leucine)
α-Ketoglutarate
Glutamate
Branched-chain
aminotransferase
(BCAT)
Branched-chain α-keto acids
(α-ketoisovalerate, α-keto-β-methylvalerate, α-ketoisocaproate)
CoA-SH
NAD+
ATP
Kinase
Branched-chain α-keto
acid dehydrogenase
(BCKDH) complex
(Active form)
Branched-chain α-keto
acid dehydrogenase
(BCKDH) complex
(Inactive form)
Phosphatase
CO2
NADH + H
+
Pi
Catabolic pathway of branched-chain amino acids.
Acyl-CoA
(Isobutyryl-CoA, α-methylbutyryl-CoA, Isovaleryl-CoA)
Succinyl-CoA
(TCA cycle)
ADP
Acetyl-CoA
Acetoacetate
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Endurance exercise stimulates energy expenditure including protein and amino acid
oxidation. The BCAA pool is maintained by degradation of muscle proteins. However,
during endurance exercise, the oxidation of BCAA may exceed the supply from proteins,
decreasing the BCAA concentrations in the blood. Leucine oxidation increases with
increased exercise intensity (Babij et al., 1983). Matsumoto et al. (2007) found that ingestion
of a single dose of 2 g BCAA plus 0.5 g arginine resulted in a significant increase in plasma
BCAA levels during three sets of 20-minute cycling at 50% maximal workload. They also
found that the increase in plasma BCAA concentrations coincided with an increase in BCAA
uptake into the muscle, whereas no increase in plasma BCAA levels or uptake of BCAA into
the muscle was observed in the placebo trial. The group observed an exercise-induced
increase in phenylalanine release from the leg during the placebo trial, indicating increased
proteolysis. However, ingestion of 2 g BCAA suppressed phenylalanine (Phe) release,
suggesting that supplementation of 2 g BCAA attenuates exercise-induced proteolysis.
Exercise has been shown to activate the BCKDH complex. Using an electrically
stimulated rat muscle contraction model, Shimomura et al. (1993) reported that increases in
muscle leucine and KIC concentrations stimulate the BCKDH complex. Furthermore, the
group also demonstrated that exercise decreases the amount of kinase bound to the BCKDH
complex (Xu et al., 2001). The result of the aforementioned findings is an increase in BCAA
catabolism during exercise. Additionally, endurance exercise training of rats decreases the
amount of BCKDH kinase in their muscles, resulting in greater activation of the BCKDH
complex (Fujii et al., 1998). The BCKDH complex of rats fed a low-protein diet was
inactivated by BCKDH kinase phosphorylation in an effort to conserve BCAA for protein
synthesis (Harper et al., 1984; Harris et al. 1990). However, when female rats were fed a
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protein-deficient diet and performed a running exercise, the activity of the BCKDH complex
was increased, suggesting that BCAA catabolism is increased during exercise even when
dietary BCAA are undersupplied (Kobayashi et al., 1999).
Fatty acid oxidation may be linked to BCAA oxidation. Administration of clofibric
acid, a hyperlipidemic drug that stimulates fatty acid oxidation also stimulates BCAA
catabolism. Long-term treatment with clofibric acid causes muscle wasting in animals (Paul
& Adibi, 1979, 1980). Long-term administration of clofibric acid decreases BCKDH kinase
activity in rats (Paul et al., 1996), which stimulates BCAA catabolism. It has been theorized
that clofibric acid exerts its long-term effects on BCKDH kinase by activation of peroxisome
proliferator-activated receptor-α (PPARα). Starvation increases fatty acid oxidation and thus
circulating concentrations of fatty acids. Because fatty acids are ligands for PPARα, BCKDH
kinase expression may be down regulated by starvation (Kobayashi et al., 2002).
Additionally, fatty acid oxidation is stimulated by exercise. Exercise-induced increases in
circulating fatty acids may therefore be associated with activation of the BCKDH,
stimulating the increase in BCAA catabolism during exercise.
Central Fatigue Theory
Two broad types of fatigue exist – central and peripheral. Central fatigue refers to
fatigue originating in the central nervous system. Peripheral fatigue is fatigue caused by
factors outside the central nervous system, primarily in the muscle. Causes of peripheral
fatigue include depletion of phosphocreatine, depletion of glycogen, accumulation of lactate,
and malfunction of the neuromuscular transmission (Åstrand and Rodahl, 1986). The causes
of central fatigue are less well known and are thought to involve primarily low blood glucose
and changes in the plasma concentration of amino acids. Gandevia (2001) describes central
13
fatigue as the state in which modifications in the central nervous system diminish motor
neuron impulse traffic to muscles. More precisely, Gandevia (2001) defines central fatigue as
“a progressive reduction in voluntary activation of muscle during exercise.”
Plasma tryptophan (Trp) is the amino acid precursor in the synthesis of 5hydroxytryptamine (5-HT) in the brain, which is involved in the control of arousal,
sleepiness, and mood (Young, 1986). According to Fernström (1990), the rate-limiting step
in the synthetic pathway of 5-HT is tryptophan crossing the blood-brain barrier. The
concentration of free-tryptophan (not bound to the plasma carrier albumin) is thought to
govern the rate of uptake in the brain. Tryptophan is the only amino acid that is bound to
albumin in the plasma. Approximately 90% of the total tryptophan in plasma is bound to
albumin; the remainder is in free form (Curzon et al., 1973). However, 5-50% of the total
tryptophan pool can be in the free form (Fernstrom and Fernstrom, 2006). The concentration
of free tryptophan in the plasma increases during, and particularly after, prolonged exercise
bouts (Lehmann et al., 1995; Blomstrand et al., 1997). Animal exercise models have
demonstrated that an increase in plasma free-tryptophan is linked to tryptophan
concentrations in the brain whereas no such relationship between plasma total tryptophan and
brain tryptophan was observed (Chaouloff et al., 1986; Blomstrand et al., 1989). Tryptophan
crosses the blood-brain barrier via the L-system, the Large Neutral Amino Acid (LNAA)
transporter (Blomstrand, 2001). Because BCAA are approximately 75% of the LNAA, and
are transported via the same transport system (Fernstrom & Wurtman, 1972), they may
compete with free tryptophan for uptake into brain. Increased free-tryptophan concentrations
lead to increased serotonin (5HT) synthesis, which is associated with lethargy, fatigue, and
decreased exercise performance (Davis et al., 2000).
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Exercise has been shown to increase the activity of 5-HT in the brain in rats (Barchas
and Freedman, 1963; Kirby et al., 1995). Administration of a 5-HT agonist during exercise
decreases running performance in rats in a dose-response manner (Bailey et al., 1992),
whereas administration of a 5-HT antagonist improves running performance (Bailey et al.,
1993). Wilson and Maughan (1992) found that administration of a 5-HT reuptake inhibitor
decreased exercise performance when human subjects cycled until exhaustion at 70% VO2
max. Increasing the ratio of free tryptophan to BCAA (fTrp:BCAA) increases the amount of
free tryptophan that can cross the blood-brain barrier and is thought to increase the synthesis
and release of 5-HT in the brain, causing central fatigue during and after prolonged exercise.
Whereas direct measurement of brain 5-HT concentrations in humans may be inherently
difficult, peripheral prolactin concentrations have been shown to be a surrogate index of
brain 5-HT activity (Yatham and Steiner, 1993).
The fTrp:BCAA significantly increases during and especially after prolonged
exercise bouts. There are several events that occur during exercise that cause fTrp:BCAA to
increase. The amount of BCAA that is taken up by the muscle increases, thereby decreasing
the plasma concentration of BCAA and ultimately increasing the free tryptophan/BCAA
ratio. Because albumin also binds and transports fatty acids in the plasma, the concentration
of plasma free fatty acids may directly influence the plasma concentration of free-tryptophan
(Knott & Curzon, 1972; Tagliamonte et al., 1971). During exercise, free fatty acids are
released from adipose tissue causing albumin to release tryptophan and bind the fatty acids,
thus increasing the amount of free-tryptophan (Curzon et al., 1973) and ultimately increasing
fTrp:BCAA. The increase in fatty acid release from adipose during exercise is caused in part
by the increase in circulating hormone levels, including epinephrine and norepinephrine
15
(Newsholme and Leech, 1983). Low levels of muscle glycogen may also influence the
release of fatty acids from adipose tissue (Widrick et al., 1993), although the mechanism is
not clearly understood.
Branched-chain amino acids supplementation may decrease sensations of fatigue and
lethargy, thereby improving exercise performance. This theory is known as the Central
Fatigue Theory (Newsholme et al., 1987).The fTrp:BCAA in the plasma may be important
for exercise-induced fatigue (Blomstrand, 2006). Exercise decreases plasma concentrations
of leucine, isoleucine, and valine, while increasing free-tryptophan. BCAA supplementation
may decrease the exercise-induced reduction of plasma BCAA, thus preventing an increase
in fTrp:BCAA. Plasma BCAA concentrations increase in response to BCAA ingestion in a
dose-dependent manner. However, plasma BCAA levels may return to baseline after one
hour of ingestion of less than one gram BCAA. Plasma BCAA concentrations maintain
elevated two hours after ingestion of more than 2 g BCAA (Hamada et al., 2005). Exercise
intensity may influence fTrp:BCAA in plasma. Strüder et al. (1998), demonstrated that the
changes in fTrp:BCAA during five hours of exercise depend on the intensity of exercise.
They observed no changes in the ratio at 50% maximum oxygen uptake, but found that the
ratio increased when participants exercised at 75% maximum oxygen uptake.
Because fTrp:BCAA in the plasma is thought to influence fatigue, according to the
Central Fatigue Theory, preventing an increase in the ratio may delay fatigue. Administration
of BCAA before or during exercise causes a rapid increase in their plasma concentrations.
Branched-chain amino acids are not taken up by the liver, thus countering the increase in free
tryptophan and preventing an increase in fTrp:BCAA and theoretically delaying fatigue.
Branched-chain amino acids may improve mood vigilance and limit central fatigue by
16
competing with free-tryptophan for uptake into the brain via the same transport mechanism
(Blomstrand et al., 1997; Pardridge and Oldendorf, 1975). Branched-chain amino acids
supplementation has been shown to decrease perceived exertion and increase endurance
performance (Blomstrand et al., 1991, 1997) and reduce brain serotonin synthesis (GomezMerino et al., 2001), consequently improving mental or physical performance or having no
ergogenic effects (Davis et al., 1999). Blomstrand et al., (1997) examined the effects of 90
mg/kg/body mass BCAA during exercise on mental fatigue and ratings of perceived exertion.
Participants performed an exercise bout the night before testing to reduce glycogen stores,
with the goal of increasing free tryptophan more quickly and subsequently causing fatigue
more quickly. Participants performed cycle ergometer exercise for 60 minutes at 70%
maximum oxygen uptake, followed by 20 minutes at their maximum. The ratings of
perceived exertion and scores of mental fatigue were lower during the BCAA trial than the
placebo trial (flavored water). Fourteen days of BCAA supplementation improved 40 km
time-trial performance of competitive cyclists (Hefler et al., 1995). Performance in triathlons
lasting three hours or longer was improved by increased fatty acid oxidation in response to
14 days of BCAA supplementation (Kreider et al. 1991).
Crowe et al. (2006) examined the effects of six weeks of leucine supplementation (45
mg/kg body mass/day; ~ 3.3 g/day) in competitive outrigger canoeists. The study population
was chosen because they commonly compete in both sprint and endurance events, providing
an excellent population on which to study the effects of chronic leucine supplementation on
endurance and power. The average training duration during the six weeks was 3.7 ± 0.3 and
3.8 ± 0.2 hours per week outrigging training plus 4.5 ± 2.1 and 4.3 ± 1.6 hours per weeks
cross training (resistance training, cycling, & jogging) for the leucine and placebo groups,
17
respectively. Six weeks of leucine supplementation increased row time to exhaustion at 7075% VO2 max (77.6 ± 6.3 to 88.3 ± 7.3 min; pre- to post-supplementation, respectively),
whereas the placebo group showed no improvement. Average ratings of perceived exertion
(RPE) of the leucine group was significantly lower during the row to exhaustion test after six
weeks of supplementation (14.5 ± 1.5 to 12.9 ± 1.4; pre- to post-supplementation,
respectively), suggesting a reduction in central fatigue, whereas the placebo group was
unchanged. Relative peak power during a ten-second arm crank test increased after six weeks
in both placebo and leucine groups. However, the increase in relative peak power after six
weeks of leucine supplementation was significantly greater, compared to placebo (6.7 ± 0.7
versus 6.0 ± 0.7 W kg-1, respectively). Row to exhaustion significantly decreased plasma
leucine (145.1 ± 4.0 to 143.2 ± 4.0 µmol), isoleucine (76.8 ± 2.3 to 75.0 ± 2.2 µmol), valine
(262.0 ± 4.6 to 250.0 ± 4.5µmol), and therefore, total BCAA concentrations (483.9 ± 10.4 to
478.2 ± 10.3 µmol). The exercise resulted in an increase in plasma free tryptophan (13.4 ±
0.8 to 14.0 ± 0.7µmol) and subsequently fTrp:BCAA (0.0280 ± 0.0020 to 0.0294 ± 0.0017
µmol). Leucine supplementation did not affect fTrp:BCAA; there was no difference in
fTrp:BCAA between the leucine and placebo groups pre- or post-supplementation before or
after exercise. These findings suggest improvements in endurance performance and upper
body power may be realized from leucine supplementation irrespective of changes in
fTrp:BCAA in plasma. The authors posit that the improvement in exercise performance from
leucine supplementation was not mediated by a reduction in central fatigue, as there was no
reduction in the ratio of fTrp/BCAA. The authors suggest the improvement in row time to
exhaustion and relative peak power output during the ten-second arm crank test from six
18
weeks of leucine supplementation may have been caused by reducing training-induced
muscle damage and enhanced recovery from training, resulting in increased performance.
Smriga et al. (2006) tested whether 5-HT release increases in areas of the brain
involved in recognition of amino acids (hypothalamus) and emotional response to stress
(amygdala) during exercise and if oral BCAA infusion moderates an exercise-induced 5-HT
release. Rats were given a 55-min acclimatization period and were orally infused with either
8 ml water or 8 ml BCAA-based solution. Rats then ran on treadmills for one hour at speeds
of either 5 m/min or 25 m/min; running at higher speeds increased circulation corticosterone
concentrations in both infusion groups. The BCAA solution increased plasma BCAA,
lowered tryptophan, and lowered fTrp:BCAA. Running at 25 m/min increased neuronal 5HT release in the lateral hypothalamus in the water-infused group; however, BCAA infusion
prevented such an increase. Serotonin release from the lateral hypothalamus 60 and 80
minutes post-exercise was significantly greater in the water-infused group than the BCAAinfused group. Treatment did not affect 5-HT release in the central nucleus of amygdala.
According to the authors, serotonin terminals projecting to the lateral hypothalamus and
amygdala are involved in the regulation of stress, food intake, and fatigue – all potentially
influenced by BCAA because BCAA infusion significantly blunted exercise-induced 5-HT
release. However, because BCAA infusion did not affect corticosterone concentrations, the
stressfulness of running was probably unaffected.
Some controversy exists on the efficacy of BCAA in regard to attenuating central
fatigue. Whereas research has shown that BCAA supplementation may attenuate exerciseinduced fatigue, some studies fail to corroborate these findings (Strüder et al., 1998;
Wagenmakers, 1992; Varnier et al., 1994). According to Fernstrom & Fernstrom (2006), the
19
general consensus is that binding of albumin to tryptophan does not influence brain uptake of
tryptophan to any meaningful level. Madras et al. (1974) tested the effects of high fat diet
consumption by rats on serum concentrations of non-esterified fatty acids (NEFA) and free
tryptophan. The diets contained 20% protein and either 0, 15, 30, or 45% fat. They found that
as fat consumption increased, serum NEFA and free tryptophan concentrations increased in a
dose-dependent manner. However, the concentration of tryptophan in the brain was not
altered, providing evidence against the regulatory action of free-tryptophan in the blood on
brain tryptophan uptake and concentration. The major critique of this study was the inclusion
of protein in the diet. The dietary protein should increase the level of LNAA in blood, which
compete for transport with tryptophan across the blood-brain barrier, effectively blunting any
potential increase in tryptophan uptake and ultimately brain tryptophan levels. Because
protein ingestion raises blood LNAA concentrations and insulin secretion lowers LNAA
concentrations, both of which may influence fTrp:BCAA, and subsequent tryptophan uptake
into the brain, Fernstrom & Fernstrom (1993) conducted a well-controlled experiment to
assess the influence of blood NEFA on brain tryptophan levels. They fed streptozotocin
diabetic rats, which cannot secrete insulin, a carbohydrate meal containing 0 or 45% fat. The
high fat diet significantly increased serum NEFA (0.56 ± 0.10 to 1.14 ± 0.11mEq/L) and
produced a two-fold increase in free tryptophan concentrations, with no effect on total
tryptophan levels two h after meal presentation. Furthermore, the ratio of freetryptophan:LNAA increased (0.030 ± 0.002 to 0.082 ± 0.011). However, despite the increase
in serum free-tryptophan and the ratio of free-tryptophan:LNAA, the concentrations of
tryptophan in the cortex and hypothalamus were unaffected. These findings question whether
the level of brain tryptophan is dependent on the concentrations of free-tryptophan.
20
Chaouloff et al. (1985) examined whether the rise in brain tryptophan during exercise
is dependent on an increase in NEFA. They found that running for two hours caused timedependent increases in plasma free tryptophan, NEFA, and consequently, brain tryptophan in
rats, leading to greater rates of 5-HT turnover, as measured by its metabolite, 5hyroxyindolacetic (5-HIAA). Administration of valine to running rats completely prevented
the exercise-induced increase in brain tryptophan and 5-HIAA. However, administration of
nicotinic acid, an inhibitor of hormone-sensitive lipase, completely abolished the runninginduced increase in plasma NEFA and plasma free-tryptophan, yet still resulted in an
increase in brain tryptophan. Chaouloff et al. (1987) suggested that increased dopamine
concentrations in the brain may inhibit 5-HT synthesis during exercise, and therefore,
attenuate fatigue. The amino acid precursor in the biosynthesis of dopamine is tyrosine (Tyr).
Tyrosine may be synthesized from phenylalanine. However, because BCAA administration
lowers the ratio of Tyr + Phe:BCAA, dopamine synthesis may be lessened when participants
are given BCAA. Because dopamine may inhibit 5-HT synthesis, additional BCAA intake
may theoretically lower the inhibition of serotonin synthesis, actually leading to fatigue.
These findings suggest that increases in brain tryptophan can occur independently of changes
in plasma NEFA and free tryptophan and BCAA supplementation may potentially dampen
the inhibitory actions of dopamine on serotonin synthesis.
Others have demonstrated that BCAA administration 70-90 minutes before short-term
exercise does not affect physical performance (Wagenmakers, 1992; Varnier et al., 1994).
Wagenmakers (1992) had participants exercise to exhaustion (about 30 minutes) in a partially
glycogen-depleted state. Varnier et al. (1994) studied the effects of administering BCAA
before a graded incremental exercise test to exhaustion, lasting approximately 40 minutes.
21
The amount of BCAA ingested during the studies by Wagenmakers et al. and Varnier et al.
was 20-30 g total, an amount that may have negative effects on exercise performance due to
a potential increase in ammonia from the muscles, causing an increase in blood levels of
ammonia, which may cause fatigue. Strüder et al., (1998) examined the effects of 21 g of
BCAA supplementation on exercise time to exhaustion. They found that BCAA
supplementation did not affect time to exhaustion, heart rate, capillary lactate, plasma insulin,
free fatty acids, glucose, serotonin, and beta-endorphin levels. Plasma ammonia increments
were higher in the BCAA trial. Branched-chain amino acids supplementation did not
attenuate the exercise-induced increase in prolactin, growth hormone, cortisol, ACTH,
epinephrine, or norepinephrine. The ratio of tryptophan:BCAA did not increase in the BCAA
trial, unlike the placebo, paroxetine, and tyrosine trials. Drive during psychometric testing
subsequent to exercise was improved in the BCAA trial. Whereas fatigue during endurance
exercise was increased by pharmacological augmentation of the brain serotonergic activity, a
reduction in 5-HT synthesis via BCAA supplementation did not affect physical fatigue.
Prolonged hyperthermia increases blood-brain barrier permeability and 5-HT
accumulation (Sharma & Dey, 1987). Increased 5-HT accumulation may augment fatigue.
Mittleman et al. (1998) examined the effects of BCAA supplementation (approximately 9 g,
females; 16 g males) on performance of a cycle ergometer ride (40% VO2 max) to exhaustion
in the heat (34°C). Exercise was performed in the heat to increase central fatigue. Mittleman
et al. (1998) found that BCAA ingestion increased ride to exhaustion time from 137 to 153
minutes, compared to placebo. However, Watson et al. (2004) observed no effect of BCAA
supplementation before and during exercise on exercise to exhaustion at 50% VO2 max in a
warm environment (30° C). Participants exercised in a glycogen-depleted state. Four 250 ml
22
aliquots of BCAA (12 g/l) were ingested in 30-minute intervals before exercise and 150 ml
aliquots were consumed every 15 minutes during exercise. BCAA ingestion did not affect
heart rate, core temperature, or weighted mean skin temperature during exercise. Whereas
ingestion of BCAA did not affect exercise capacity in the heat, BCAA ingestion did lower
fTrp:BCAA in plasma, compared to placebo. This finding refutes the Central Fatigue Theory.
Treatment had no effect on blood glucose or lactate concentrations. Ingestion of BCAA did
elevate plasma ammonia during exercise, whereas plasma ammonia concentrations did not
change in the placebo group. The findings of Watson et al. (2004) suggest that exercise
capacity in the heat is not hindered by greater ammonia concentrations and is not influenced
by fTrp:BCAA in the plasma.
If administration of BCAA decreases fTrp:BCAA and consequently attenuates central
fatigue, one could hypothesize that administration of tryptophan would increase fTrp:BCAA
and augment central fatigue. Farris et al. (1998) found that infusion of tryptophan to horses
decreased running time to exhaustion at 50% VO2 max (87 vs. 122 minutes for tryptophan
and saline infusion, respectively). Additionally, Weicker and Strüder (2001) found that
endurance-trained male cyclists reached time to exhaustion during cycle ergometry
significantly earlier when administered 20 mg paroxetine (selective serotonin reuptake
inhibitor, SSRI; 131 ± 53 min) than placebo or 14 g BCAA (157 ± 53 and 152 ± 41 min,
respectively). After the exercise bout, participants were given a cognitive test (KL-test,
Düker 1976). Lower scores for subjects’ drive or motivation (95.1 ± 34.4 and 108.9 ± 43.3
for SSRI and placebo, respectively) and a greater percentage of incorrect responses (4.8 ± 3.9
and 7.8 ± 4.8 for SSRI and placebo, respectively) were observed in the paroxetine trial versus
23
the placebo trial. The authors suggest that the reduction in physical performance observed in
the SSRI trial may have been caused by a reduction of 5-HT in the synaptic cleft.
Carbohydrates, BCAA, and Central Fatigue
Consumption of carbohydrates during prolonged exercise may improve performance
by maintaining blood glucose levels and central nervous system function. Carbohydrate
consumption may decrease central fatigue by attenuating the exercise-induced rise in plasma
free fatty acids (Wright et al., 1991; Widrick et al., 1993; McConell et al., 1999), the
subsequent displacement of tryptophan from albumin, and consequently plasma free
tryptophan (Davis et al., 1992). Consumption of carbohydrate stimulates insulin secretion,
which is known to inhibit lipolysis, and stimulate lipogenesis, thereby decreasing plasma free
fatty acid levels. However, during exercise that lasts two to three hours, an increase in plasma
free fatty acids is observed, despite consumption of carbohydrates (Wright et al., 1991; Davis
et al., 1992; McConell et al., 1999). The increase in plasma free fatty acids is likely caused
by low levels of glycogen, blood glucose or insulin. Because prolonged exercise lowers
muscle glycogen levels and stimulates the uptake of BCAA into muscle for oxidation
(Blomstrand and Newsholme 1992), fTrp:BCAA increases during exercise. Because
fTrp:BCAA increases during exercise, the ratio of fTrp:LNAA (large neutral amino acids)
also increases during exercise, leading to an increase in cerebral total tryptophan (free and
bound) and brain 5-HT synthesis. According to Weicker and Strüder (2001), an overproportional release of tryptophan from albumin occurs and, as a result, higher levels of 5HT are synthesized in the brain during states of glycogen depletion. Nutritional interventions
using carbohydrate and/or BCAA may therefore cause alterations in the manifestation of
central fatigue during exercise.
24
Several studies have investigated the effects of BCAA plus carbohydrate intake on
physical performance. Divergent findings exist on the additional benefit of BCAA
supplementation over carbohydrate ingestion alone. Welsh et al. (2002) reported that
carbohydrate ingestion increases physical and mental function during intermittent exercise.
Most have observed no additional benefit of BCAA over carbohydrate ingestion alone
(Blomstrand et al., 1995; van Hall et al., 1995; Madsen et al., 1996; Davis et al., 1999).
However, Blomstrand et al. (1991) found that slower runners given BCAA improved
performance in a marathon run. The results obtained by Davis et al., (1999) do not support
the hypothesis of an added benefit of BCAA supplements on fatigue. They found that both
carbohydrate one hour before (5 ml/kg; 18% carbohydrate) and during (2 ml/kg; 6%
carbohydrate) intermittent exercise to fatigue (shuttle running; walking, sprinting, and
running) and carbohydrate plus 7 g BCAA increased time to exhaustion compared to a
flavored water placebo trial. The addition of BCAA did not yield any added physical
performance benefit. Plasma insulin and glucose were higher and FFA were lower in the
carbohydrate and carbohydrate plus BCAA trials than the placebo trial.
Kaastra et al. (2006) tested the potential additive effects of protein and leucine
ingestion with carbohydrates on insulin secretion and glucose disposal during recovery from
exercise in endurance-trained males. They observed that ingestion of a protein hydrolysate
with carbohydrates and ingestion of a protein hydrolysate, free leucine, and carbohydrates
increased insulin secretion more than ingestion of carbohydrates alone (108 ± 17 and 190 ±
33 % greater, respectively), but did not affect plasma glucose disposal. Cheuvront et al.
(2004) examined whether BCAA supplementation affects performance when participants
were hypohydrated by 4% body mass and were glycogen-depleted. They found that BCAA
25
when combined with carbohydrate does not provide any additive benefit on physical
performance over carbohydrate ingestion alone under conditions in which central fatigue
may have been exacerbated by alterations in serotonergic function. However, BCAA
ingestion resulted in an increase in plasma BCAA, and a decrease in the ratios of total
Trp:BCAA and total Trp:LNAA, compared to a carbohydrate placebo. Neither carbohydrate
nor carbohydrate plus BCAA affected plasma FFA or prolactin concentrations. Interestingly,
participants’ heart rates were higher during the BCAA plus carbohydrate trial than the
carbohydrate placebo trial. No treatment effect was observed for ratings of perceived
exertion, thermal comfort, mood as measured by the POMS, or any test of cognitive function
(four-choice reaction time, visual vigilance, match-to-sample, repeated acquisition, and
grammatical reasoning).
Ingestion of carbohydrates may reduce the uptake of tryptophan into the brain.
Blomstrand et al. (2005), observed that during prolonged exercise (three hours of cycle
ergometry at 200 ± 7 Watts), carbohydrate ingestion (6%) attenuated the uptake of
tryptophan into the brain, as measured by the difference of radial artery and jugular venous
blood concentrations multiplied by plasma flow. In the placebo (flavored water) trial,
approximately 106 µmol (22 mg) of tryptophan was taken up by the brain during exercise,
which began 30 minutes into the three hour exercise bout and continued for the remaining 2.5
hours. No uptake of tryptophan was observed in the carbohydrate trial. The arterial
concentration of free tryptophan increased to a greater extent during the placebo trial,
compared to the carbohydrate trial (12 ± 1 to 20 ± 2 µ mol L-1 during the placebo trial,
compared to 14 ± 1 µ mol L-1 at the end of the carbohydrate trial). The concentration of total
tryptophan (free and bound) increased during the first 30 minutes of both trials, but returned
26
to baseline at 180 minutes of exercise. Branched-chain amino acids were taken up by the
brain in both trials. No correlation between plasma free tryptophan or fTrp:BCAA in blood
and the uptake of tryptophan into the brain were found. However, in exercising animal model
studies, a proportionate increase in plasma free tryptophan (not total) and increase in brain
tryptophan has been observed (Chaouloff et al., 1986).
BCAA and Anaerobic Performance
The effects of BCAA supplementation on anaerobic exercise performance have not
been extensively studied. Acute ingestion of leucine (200 mg/kg body mass) before a
maximal anaerobic running exercise session until exhaustion had no effect on performance of
the test, compared to placebo (Pitkänen et al., 2003). Serum leucine was higher in the leucine
supplementation group than the placebo group both ten minutes before and ten minutes after
exercise and was higher after the exercise session in response to leucine supplementation.
Isoleucine and valine concentrations were lower after exercise than before in the leucine
group. The group also tested the effects of acute ingestion of leucine (100 mg/kg body mass)
before and during a strength exercise session. Leucine ingestion did not affect performance
of a counter movement jump. The effect on leucine ingestion before and after a strength
exercise session on serum amino acid concentrations was similar to that of the maximal
anaerobic running session. Serum leucine concentrations were higher in the leucine
supplemented group before and after exercise, compared to placebo. Serum leucine
concentration decreased after exercise in the placebo group, whereas leucine supplementation
prevented an exercise-induced decrease in serum leucine. Serum isoleucine and valine
decreased more after exercise in the leucine group than the placebo group. The participants in
this study were competitive male power athletes. It is possible that their performance on
27
anaerobic running tests or counter movement jump tests are already rather high, therefore
acute ingestion of leucine is unlikely to provide an ergogenic benefit. Additionally, counter
movement jumping is a high-skill power movement. Perhaps acute ingestion of leucine may
improve power output in less skill-intense tests.
Crowe et al. (2006) found that relative peak power during a ten-second arm crank test
increased after six weeks of supplementation and cross training in both placebo and leucine
treatment groups. However, the increase in relative peak power after six weeks of leucine
supplementation was significantly greater compared to placebo (6.7 ± 0.7 versus 6.0 ± 0.7 W
kg-1, respectively). Leucine supplementation did not influence total work performed in the
ten-second arm crank test. Vukovich et al. (1992) found that seven days of supplementation
with an amino acid mixture (0.8 g/kg body mass; rich in BCAA) did not affect power output
during a Wingate test; however, a trend for lower percent fatigue was observed in the amino
acids trial, compared to placebo (44.82 ± 2.21 and 51.45 ± 2.98% for amino acids and
placebo, respectively). These findings question the efficacy of BCAA supplementation on
anaerobic exercise performance.
BCAA and Buffer Capacity
Branched-chain amino supplementation may influence lactate accumulation in the
blood during exercise. Lactate in the blood increases when the rate of lactate production
exceeds the rate of lactate removal. Pyruvate is the end-product of glycolysis. The four fates
of pyruvate are 1) enter the Kreb’s cycle as Acetyl CoA, 2) transaminated to alanine,
catalyzed by the enzyme glutamate pyruvate transaminase (GPT), 3) transported to the liver
for gluconeogenesis, and 4) reduced to lactate, catalyzed by the enzyme lactate
dehydrogenase. Increased BCAA availability has been shown to increase the transamination
28
of pyruvate to alanine (Odessey et al., 1974), thereby decreasing lactate formation (Mole et
al., 1973), potentially ameliorating the onset of fatigue. Vukovich et al. (1992) found that
seven days of supplementation with an amino acid mixture (0.8 g/kg body mass; rich in
BCAA) significantly lowered participants’ blood lactate concentrations when they exercised
at intensities greater than 100% VO2 max, but did not affect blood lactate concentrations at
intensities less than 100% VO2 max. They also found that concentrations of blood alanine
were higher at all exercise intensities (40, 60, 80, 100, & 120% VO2 max). Whereas amino
acid supplementation did not affect power output during a Wingate test, a trend for lower
percent fatigue was observed in the amino acids trial. Muscle lactate and muscle glycogen
concentrations were lower after the Wingate test in the amino acid trial than the control.
Seven days of amino acid supplementation significantly increased pre-exercise GPT activity
by 47%, but had no effect on the activity of citrate synthase (CS), a Kreb’s cycle enzyme.
Maresh et al. (1991) observed lower blood lactate concentrations after a Wingate test
when participants consumed an amino acid supplement. They posited the lower lactate
accumulation was due to increased transamination of pyruvate, increased oxidation of amino
acids, decreased lactate efflux from muscle to blood, reduced intracellular ammonia
accumulation, or increased clearance of lactate by the liver. The findings of Vukovich et al.
(1992) on increased GPT activity in response to amino acid supplementation support the
theory of increased transamination. The authors suggest the increase in GPT activity after
amino acid supplementation is likely due to increased amino acids available as substrate for
GPT production, increasing the amount of the enzyme, which increase its activity.
Furthermore, BCAA are the primary amino acids involved in the transamination of 2oxoglutarate to glutamate, the reaction immediately preceding that catalyzed by GPT.
29
Increasing the activity of GPT may increase the transamination of pyruvate to alanine,
decreasing the reduction of pyruvate by lactate dehydrogenase to lactate, potentially
decreasing the amount of lactate in the blood and muscle (Mole et al., 1973).
Branched-chain amino acid supplementation may improve muscle buffering capacity.
Because most athletes and the general population combine aerobic and anaerobic training,
Vukovich et al. (1997), studied the effects of six weeks of BCAA plus glutamine and
carnitine supplementation (2.9 g/day) on untrained people performing combined aerobic and
anaerobic training. The authors also studied the effects of seven days of BCAA
supplementation without exercise on muscle and blood lactate accumulation. They found that
seven days of supplementation without exercise training did not affect peak oxygen
consumption, power output associated with onset of blood lactate accumulation (4 mM;
OBLA), and blood lactate concentrations. Time to ride to exhaustion at 120% VO2 peak,
muscle lactate, pH, and buffer capacity after the ride to exhaustion were not affected by
seven days of BCAA supplementation. Six weeks of combined aerobic and anaerobic
training increased peak oxygen consumption, power output at OBLA, and ride time to
exhaustion at 120% VO2 max. There was no additional benefit from BCAA supplementation.
Blood lactate concentration at VO2 peak was not affected by training or supplementation. In
vivo buffer capacity increased significantly after two weeks of training in the BCAA group;
this increase was not observed in the control group. Muscle proteins have been shown to be
involved in the increased muscle buffer capacity associated with training (Sahlin &
Henricksson, 1984). The authors posit the BCAA supplementation may have increased
protein anabolism or decreased protein catabolism, resulting in greater muscle protein, and
thereby improving muscle buffer capacity. Vukovich et al. (1997) suggest that a threshold of
30
greater than 2.9 g per day BCAA supplementation may be needed to attenuate exerciseinduced increases in blood and muscle lactate accumulation.
Alanine, a gluconeogenic precursor, has been shown to increase the secretion of
glucagon (Brooks and Fahey, 1985). Glucagon stimulates gluconeogenesis and
glycogenolysis. Vukovich et al. (1992), observed lower muscle glycogen content after a
Wingate test after seven days of amino acid supplementation. They suggested the increased
glycogen use may have been due to increased glucagon secretion in response to greater blood
alanine concentrations, although they did not measure glucagon. Amino acid
supplementation may potentially decrease long-term endurance performance if in fact
glycogenolysis is stimulated, increasing the probability of athletes becoming glycogendepleted. However, if alanine stimulates glucagon secretion, which in turn stimulates
gluconeogenesis, blood glucose levels may be better maintained during prolonged exercise in
response to amino acid supplementation, decreasing the chance of athletes experiencing
hypoglycemia. Whether glucagon secretion in response to amino acid supplementation
mediates endurance performance has yet to be elucidated.
Responders to BCAA Supplementation
There may be responders and non-responders to BCAA supplementation (Maresh et
al. 1991, 1994). Maresh et al. (1991, 1994) found participants who were more physically
active – expending more than 1000 kcals per day – had lower plasma lactate concentrations
after a 30-second Wingate test than those reporting daily expenditure of less than 500 kcals
after consuming ~ 7.6 g amino acids per day for 14 days. Vukovich et al. (1997) corroborate
this theory of responding to amino acid supplementation. They found that seven days of
supplementation did not influence blood or muscle lactate concentrations in sedentary
31
participants expending fewer than 500 kcals per day. Thus, amino acid supplementation may
be more likely to lower blood and muscle lactate accumulation during exercise in individuals
who have a greater energy demand from increased physical activity.
Need for Additional BCAA Intake
People beginning an exercise program may have increased demands for BCAA
consumption. When untrained people begin a physical activity program, they may be in
negative nitrogen balance for up to ~ 21 days (Gontzea et al., 1975), indicating greater
protein degradation than synthesis, or net protein loss. This suggests people beginning an
endurance training program may have greater dietary protein requirements. The negative
nitrogen balance has been improved to a greater extent with ingestion of 1.5 g protein/kg
body mass per day than 1.0 g protein/kg mass (Gontzea et al., 1974). These findings are
corroborated by Vukovich et al. (1997). They observed two weeks of BCAA
supplementation resulted in less 24-hour urea excretion after the ride to exhaustion at
120%VO2 max than two weeks of placebo. This suggests that two weeks of BCAA
supplementation attenuates protein degradation during the early stages of combined aerobic
and anaerobic exercise program.
BCAA and Cognition
The synthesis of catecholamines (dopamine, epinephrine, and norepinephrine) in the
brain is dependent on the availability of their amino acid precursors tyrosine (Tyr) and
phenylalanine (Phe). Administration of BCAA lowers the plasma ratio of Tyr + Phe:BCAA
and therefore, may decrease the synthesis of catecholamines and lead to cognitive changes
consistent with impaired dopamine activity. Scarnà et al. (2005) gave participants an amino
acid mixture comprised of 60 g BCAA (valine 18g, isoleucine 18 g, and leucine 24 g) plus 2
32
g tryptophan or placebo. The BCAA/Trp mixture resulted in higher levels of BCAA and
tryptophan in the plasma and decreased ratios of Tyr + Phe:BCAA and Trp:BCAA. The
absolute levels of tyrosine and phenylalanine decreased, perhaps due to increased protein
synthesis stimulating their uptake, caused by ingesting the amino acid mixture. The amino
acid mixture also resulted in higher levels of prolactin. Prolactin is a hormone synthesized
and secreted from the anterior pituitary. Dopamine inhibits prolactin synthesis. The increase
in plasma prolactin is consistent with lower dopamine levels caused by administration of
BCAA. Participants receiving the Trp/BCAA treatment were significantly more accurate (96
± 32 vs. 86 ± 29% for Trp/BCAA and placebo, respectively) and faster to respond during a
pattern recognition memory test (1645 ± 579 vs. 2163 ± 1071 ms for Trp/BCAA and
placebo, respectively) than their counterparts receiving the placebo, for which the authors
have no clear explanation. Participants in the Trp/BCAA group were also more likely to
make risky choices on an experimental gamble scenario, suggesting alterations in the
catecholamine system influence the capacity to choose actions associated with risks and
rewards. Interestingly, participants receiving the Trp/BCAA mixture reported feeling more
fatigue at both 270 and 330 minutes and less vigorous 330 minutes after ingestion, compared
to those consuming the placebo, as measured by the Profile of Mood States questionnaire.
The fact that participants reported feeling more fatigued after receiving the Trp/BCAA
mixture is puzzling because fTrp:BCAA decreased, and according to the Central Fatigue
Theory, one would expect fatigue to be attenuated in such a condition. The authors
hypothesize the increase in fatigue may be caused by a decrease in catecholamine
availability.
33
Branched-chain amino acids may either improve or prevent loss of cognitive
performance after endurance exercise (Hassmén et al., 1994). Participants given BCAA
during a 30-km cross-country race improved their performance by 3-7% in the different parts
of the word-color test (words, colors and color words) after exercise, whereas no difference
was observed before and after the race in participants given a carbohydrate placebo.
Performance in shape-rotation and figure-identification tasks decreased after the race in
participants receiving placebo (25 and 15%, respectively), whereas performance of these
tasks was maintained in participants receiving BCAA. The authors suggest BCAA
supplementation is more likely to maintain cognitive performance of more complex tasks
than less demanding ones after endurance exercise. The effects of amino acid
supplementation on exercise-mediated cognitive function are not fully understood and more
research in the area is needed.
BCAA and Physical Activity
Branched-chain amino acid supplementation may influence free-living voluntary
physical activity. Through a series of studies using exercising rats, Smriga et al. (2006) tested
the hypothesis that greater dietary intakes of BCAA may affect voluntary physical activity
behavior. In one study, adult male rats housed in cages equipped with a running wheel were
given one of two diets: standard laboratory diet fortified with 20 g/kg BCAA or control,
standard laboratory diet with 20 g/kg glutamine. The group receiving the control diet ran the
same distance each day; however, the group receiving the BCAA-fortified diet gradually
increased their running distances. After day four, the BCAA group was running significantly
greater distances than the control group. Whether similar results would be observed in human
studies is unknown as behavior change is likely more complex in humans. In a second study,
34
Smriga and colleagues examined the effect of exercise on ad libitum BCAA consumption.
Rats were housed in cages containing running wheels, a pellet dispenser, and two fluid
dippers. One dipper contained water, the other a BCAA-based amino acid solution (15.2
mmol/L leucine, 9.9 mmol/L isoleucine, 11.1 mmol/L valine, 16.6 mmol/L glutamine, and
13.9 mmol/L arginine). They found that food intake was not related to running distance. Rats
running > 1500 m during the dark period (1900-700 h) consumed significantly more BCAA
solution than water. The authors concluded from these studies that BCAA intake and
physical activity in rats are related. When the physical activity of rats increased, so did their
voluntary consumption of BCAA and when rats were fed greater amounts of BCAA, they
voluntarily increased their physical activity. One should note, in the second study, the fluid
dippers contained either water or BCAA. These two treatments are not isocaloric, so it could
be posited that rats chose to drink more BCAA solution to increase their caloric intake to
compensate for greater energy expenditure. A follow-up study is warranted to distinguish the
contribution of BCAA per se, or their caloric content. Furthermore, a study examining the
effects of BCAA supplementation on free living voluntary physical activity in humans is
highly warranted.
Leucine Supplementation and Obesity
Branched-chain amino acids may mediate body mass regulation and blood lipid
profiles. Donato et al. (2006) found that leucine supplementation during caloric restriction
increased fat loss and improved protein synthesis in muscle and liver in rats. Zhang et al.
(2007) conducted a study in which they fed mice one of four diets for 14 weeks: regular
rodent chow, regular rodent chow supplemented with leucine, high-fat diet, or high-fat diet
supplemented with leucine. Leucine supplementation increased plasma leucine
35
concentrations, but decreased overall plasma amino acids levels, suggesting a decrease in
protein catabolism. Leucine supplementation significantly attenuated the body mass gain
(32% less) and increased adiposity (25% less) observed in mice consuming high-fat diets,
despite similar total caloric intakes between the high-fat diet groups. The mice consuming the
high-fat diet supplemented with leucine had greater total energy expenditure than the high-fat
diet group. Furthermore, the respiratory exchange ratio (RER) was lower in mice consuming
the leucine-supplemented high-fat than those consuming the high-fat diet, suggesting greater
fat oxidation from leucine supplementation. Additionally, increasing leucine intake improved
glucose tolerance (34.2 ± 7.6 vs. 67.7 ± 11.7 homeostasis model assessment of insulin
resistance index for the leucine and control groups, respectively) and insulin sensitivity in
mice fed high-fat diets. Forward-stepwise regression indicated that the glucose-lowering
effect of leucine was independent of the decrease in adiposity. Leucine supplementation for
14 weeks decreased total cholesterol 27% and LDL-cholesterol 53% in high-fat diet mice.
However, leucine had no effect on HDL-cholesterol, total triglycerides, or free fatty acid
concentration. Leucine had no noteworthy effect on mice consuming the normal rodent chow
diets.
Supplementation of BCAA (76% leucine) during periods of caloric restriction may
result in greater body weight mass loss, decrease in percent body fat, and preferential
reduction in abdominal visceral adipose tissue, as observed in competitive wrestlers
(Mourier, et al., 1997). Additionally, during periods of caloric restriction, no change in
aerobic (VO2 max) or anaerobic (Wingate test) capacities and muscular strength were
observed. These results suggest BCAA supplementation during periods of moderate caloric
36
restriction augments preferential visceral adipose loss while maintaining a desirable, high
level of performance.
Lateral hypothalamic 5-HT activity is inversely related to food intake (Leibowitz et
al., 1990); decreases in hypothalamic 5-HT activity increases food intake. Branched-chain
amino acid supplementation may decrease neural 5-HT activity, potentially mediating
voluntary food intake. Cota et al., (2006) found that central administration of leucine to the
third ventricle of the brain decreased food intake and body weight. However, based on the
findings of their behavioral studies in which food intake was unaffected by ad libitum BCAA
access, Leibowitz et al., (1990) suggest BCAA may not be centrally involved in influencing
feeding behaviors.
BCAA and Muscle Recovery
Some studies have shown that BCAA supplementation may enhance recovery after
strenuous exercise. Sugita et al. (2003) had male students perform a session of eccentric
exercise training. During ten days of recovery, participants consumed an amino acid mixture
(5.6g; ~30% by weight BCAA, 14% glutamine, 14% arginine, threonine, lysine, proline,
methionine, histidine, phenylalanine, and tryptophan plus vitamins and minerals) or
isocaloric placebo (vitamins and minerals plus carbohydrate) twice per day. Ingestion of the
amino acid mixture accelerated recovery from eccentric exercise. Whereas force production
decreased in both groups after eccentric exercise, maximal isometric force production during
elbow extension was greater in the amino acid group than the placebo group. Interestingly,
no treatment effect was observed for elbow flexion.
The group also conducted a dose-response study to examine the effective dose for
reducing training-induced muscle damage (Ohtani et al., 2001). Middle- and long-distance
37
college male track athletes consumed 2.2, 4.4, and 6.6 g/day of the previously described
amino acid mixture for one month. A one-month washout period between treatments was
given. Level of training was maintained constant during the six months by altering running
distance and intensity. Blood samples were taken in a fasted state one day after no physical
activity before and after each one-month treatment period. The 2.2 g/day dose did not affect
blood measures of damage and oxygen-carrying capacity. The 4.4 g/day dose increased
serum albumin and reduced serum iron and blood lactate concentrations. The 6.6 g/day dose
produced significant changes in oxygen-carrying capacity, physical condition, and blood
markers of muscle damage. Blood glucose concentrations were greater after one month
supplementation of 6.6 g/day amino acid mixture (88 ± 1 and 93 ± 1 mg/dl pre- and post-test,
respectively), suggesting increased glycogenesis, according to the authors. Red blood cell
count, hemoglobin concentration, and percent hematocrit increased after one month
supplementation of 6.6 g/day, suggesting an increase in hematopoiesis and oxygen-carrying
capacity. One month of 6.6 g/day amino acid supplementation resulted in higher selfassessed physical condition scores, greater serum albumin concentrations, lower CPK, or
LDH. The lower CPK activities suggest faster recovery from training-induced inflammation.
Coombes et al. (2000) found that 40 g BCAA (approximately 550 mg/kg body mass) reduced
muscle damage after prolonged endurance exercise. The exercise-induced muscle protein
breakdown may be attenuated by BCAA supplementation before and during prolonged
exercise (MacLean et al., 1994; MacLean et al., 1996), mediating exercise-induced muscle
damage.
38
Safety of BCAA Consumption
High doses of BCAA may have negative effects. The recommended daily protein
intake is 0.8 g/kg body mass for adults and 1.2 to 1.8 g/kg body mass for athletes
(Jeukendrup & Gleeson, 2004). High daily protein intake (> 3 g/kg body mass) may cause
dehydration, increased blood lipoprotein levels, and potentially kidney failure (Gleeson,
2005). Plasma ammonia concentrations may increase when participants are given large
amounts of BCAA (20-30 g) before or during exercise (Wagenmakers, 1992; MacLean and
Graham, 1993; van Hall et al., 1995; MacLean et al., 1994). However, others have observed
no increase in ammonia after BCAA administration before or during exercise (Varnier et al.,
1994; Blomstrand et al., 1997; Mittleman et al., 1998). Smaller doses of BCAA (7-10 g; 100
mg/kg/body mass) do not stimulate release of ammonia from muscles (Blomstrand et al.,
1997). According to Gleeson (2005), daily intakes of 10-30 g BCAA do not appear to be
harmful. More precisely, daily intakes of up to 450 mg/kg body mass of BCAA appear to be
well tolerated in healthy adults (Gleeson, 2005). A dose-response study of BCAA
supplementation dose, measures of physical performance, alterations in the ratio of plasma
fTrp/BCAA, and plasma ammonia concentrations is warranted (Crowe et al., 2006).
Supplementation of BCAA for one month does not appear to have adverse effects on
liver function. Ohtani et al. (2001) found that one month of 6.6 g/day amino acid
supplementation resulted lower glutamate-oxaloacetate transferase (GOT) activity.
Supplementation did affect activities of GPT, or γ-glutamyltranspeptidase (GTP). GOT,
GPT, and γ-GTP are markers of liver function; greater activities suggest disturbance of liver
function and or states of liver disease. In fact, BCAA supplementation to liver cirrhosis
patients decreased the activity of alkaline phosphatase, another marker of liver damage (Suga
39
et al., 1994). These findings suggest no adverse liver function consequences result from
amino acid supplementation. However, controlled long-term studies examining the effects of
amino acids supplementation on kidney and liver function are highly warranted.
BCAA Summary
The three branched-chain amino acids – leucine, isoleucine, and valine have been
shown to positively affect several measures in human and animal studies. The efficacy of
BCAA supplementation on exercise performance has been posited to depend on several
factors including the mode of administration (Verger et al., 1994), the nutritional status of
recipients (Calders et al., 1997), use in conjunction with carbohydrates (Blomstrand, 2001),
and the length and intensity of exercise (Strüder et al. (1998). Blomstrand (2001) argues that
the benefits of BCAA use are likely to be greater during activities in which the central
component of fatigue plays a considerable role, such as exercise in the heat or during a
competitive race. Branched-chain amino acids may limit central fatigue by competing with
free-tryptophan for uptake into the brain via the same transport mechanism (Blomstrand et
al., 1997; Pardridge and Oldendorf, 1975). Branched-chain amino acids supplementation has
been shown to decrease perceived exertion and increase endurance performance (Blomstrand
et al., 1991, 1997) and reduce brain serotonin synthesis (Gomez-Merino et al., 2001;Smriga
et al. 2006). Whereas research has shown that BCAA supplementation may attenuate
exercise-induced fatigue, some studies fail to corroborate these findings (Strüder et al., 1998;
Wagenmakers, 1992; Varnier et al., 1994). Branched-chain amino acid supplementation may
improve muscle buffering capacity (Vukovich et al. (1997). Increased BCAA availability has
been shown to increase the transamination of pyruvate to alanine (Odessey et al., 1974),
thereby decreasing lactate formation (Mole et al., 1973), potentially ameliorating the onset of
40
fatigue. Vukovich et al. (1992) found that seven days of supplementation with an amino acid
mixture (0.8 g/kg body mass; rich in BCAA) significantly lowered participants’ blood lactate
concentrations when they exercised at intensities greater than 100% VO2 max. Infusion of
BCAA, particularly leucine, has been shown to increase protein synthesis (Alvestrand et al.,
1990) and decrease protein breakdown (Louard et al., 1990; Nair et al., 1992), potentially
resulting in net protein accretion.
Branched-chain amino acid supplementation may have health benefits. Consumption
of BCAA influences free-living voluntary physical activity (Smriga et al. (2006). Leucine
supplementation for 14 weeks improved glucose tolerance, lowered total cholesterol, and
LDL-cholesterol in high-fat diet mice (Zhang et al., 2007). Supplementation of BCAA (76%
leucine) during periods of caloric restriction may result in greater body weight mass loss,
decrease in percent body fat, and preferential reduction in abdominal visceral adipose tissue,
as observed in competitive wrestlers (Mourier, et al., 1997). Finally, supplementation of
BCAA (6.6 g/d) for one month does not appear to have adverse effects on liver function
(Ohtani et al., 2001). More research is needed on the long-term effects of consuming high
protein diets or amino acids supplements.
Carbohydrate mouth rinse
Carbohydrate ingestion before and during short-term and intense exercise sessions
has been shown to delay fatigue and improve exercise (Bergstrom et al., 1967; Coggan &
Coyle, 1987; Foskett et al., 2008). The mechanism underlying reported enhancement is not
well understood. One theory suggests carbohydrate feeding may enhance rates of
carbohydrate oxidation. However, for short-term, high-intensity exercise bouts, this theory
may be unlikely as only 5 to 22 grams of exogenous carbohydrates are oxidized in the first
41
hour of exercise, minimally contributing to total carbohydrate oxidation rates, and an amount
possibly too small to significantly enhance exercise performance (Jeukendrup et al., 1997).
Additionally, some have reported no difference in rates of carbohydrate oxidation during
high intensity exercise when given carbohydrates or placebo (Neufer et al., 1987; Below et
al., 1995). Furthermore, muscle glycogen contributes much more carbohydrates for oxidation
than does the liver or blood during one hour of exercise. Others have demonstrated that
carbohydrate feeding before and during exercise bouts does not enhance performance
(Palmer et al., 1998; Clark et al., 2000). Carbohydrate ingestion has been shown to improve
performance when muscle glycogen stores become depleted, which usually occurs during
longer duration exercise bouts (Coyle et al., 1986). Hawley et al. (1997) demonstrated that
after one hour of all-out cycling, adequate glycogen remains in the muscle. Whereas no clear
metabolic explanation exists for improved exercise performance when given a carbohydrate
solution, the general consensus is that carbohydrate feeding likely improves exercise
performance.
Prolonged exercise bouts deplete muscle glycogen content, which can decrease
exercise performance. Feeding carbohydrate before and during exercise may improve
performance and delay fatigue. Ingestion of carbohydrate increases glucose metabolism in
the muscle, leading to a slower rate of glycogen depletion (Tsintzas et al., 1996). However,
carbohydrate ingestion has been shown to improve exercise performance independent of
muscle glycogen sparing (Coyle et al., 1986). Others have shown carbohydrate ingestion
does not influence carbohydrate oxidation, muscle metabolism, or exercise performance
while exercising to fatigue (McConell et al., 2000). Furthermore, a relatively small
percentage (26%) of total carbohydrate ingested enters the peripheral circulatory system
42
during high-intensity endurance exercise (McConell et al., 2000). However, several studies
have shown that carbohydrate ingestion improves exercise performance without influencing
muscle metabolism (Anantaraman et al., 1995; Powers et al., 1990)
Whereas it is generally accepted that carbohydrate ingestion improves exercise
performance during prolonged exercise bouts in which glycogen depletion may occur, the
effects of carbohydrate ingestion on exercise performance in shorter-term bouts are less well
understood. Neufer et al. (1987) found that carbohydrate ingestion (45g) by well-trained
cyclists increases the total amount of work performed in 15 minutes by approximately 10%
after cycling for 45 minutes at 77% VO2 max. The mechanism for the improvement in shortterm exercise bouts from carbohydrate ingestion has yet to be fully elucidated. Some have
suggested that greater rates of carbohydrate oxidation may be implicated in increased
performance; however, it has been demonstrated that no difference in carbohydrate oxidation
rates exists during 45-60 minutes of exercise between a carbohydrate ingestion trial and
placebo (Neufer et al., 1987; Below et al., 1995).
Central or non-metabolic factors may mediate improvements in performance in
response to carbohydrate consumption. Carter et al. (2004a) found that whereas glucose
infusion (one g/min) resulted in abundant available circulating glucose, infusion did not
improve performance in a simulated 40 km time trial bout. The group suggests that oral
carbohydrate feeding may exert its effects through a central mechanism, improving motor
drive or motivations, mediated by receptors in the mouth or gastrointestinal (GI) tract.
To test the hypothesis that receptors in the mouth may stimulate a central drive
mechanism, Carter et al. (2004b) had participants rinse the mouth with either a carbohydrate
solution (6.4 % maltodextrin) or water and without swallowing, spit out the treatment, thus
43
eliminating potential stimulation from GI tract receptors to induce a metabolic action.
Participants were asked to complete a given amount of work (0.75 x W x 3600) as quickly as
possible. This time trial procedure is highly reproducible when performed in this way using
participants who are experienced in this type of exercise (Jeukendrup et al., 1996).
Participants were given a 25-ml bolus of carbohydrate solution or water every 12.5% of the
time trial completed, which was rinsed around their mouths for approximately five seconds
and spat into a bowl held by an investigator. Participants were informed that both treatments
had been previously reported to improve exercise performance. Time to complete the
performance trial was significantly less (2.9%) when participants rinsed with carbohydrate
compared to water. Power output was therefore greater when participants rinsed with
carbohydrate. Furthermore, mean power output was greater during the first three-fourths of
the trial when receiving the carbohydrate rinse than the water rinse. There were no
differences in heart rate, ratings of perceived exertion, or body mass changes between the
treatment groups. Five of the nine participants were unable to distinguish between the
treatments. The four who correctly identified treatment reported a different feel or viscosity
between the carbohydrate and water solutions.
The mechanism for time trial improvement when rinsing with or ingestion of a
carbohydrate solution is unclear. There may be “nonmetabolic” or central mechanisms
involved. One hypothesis is that ingestion of carbohydrate may increase circulating glucose
concentrations, thereby improving glucose availability to the brain, enhancing central
nervous system functioning. When rinsing with carbohydrates, increased glucose delivery to
the brain is not likely. A second hypothesis is that ingestion or rinsing with carbohydrates
activates receptors in the mouth that stimulate an afferent pathway to the brain. Carter et al.
44
(2004b) suggested that oral cavity receptors may stimulate reward centers of the brain.
Glucose receptors are well known to exist in the gut and brain of humans. Additionally,
various taste receptors exist in the oral cavity of humans, including those that sense sweet
foodstuffs on the tongue. However, because maltodextrin is not a sweet carbohydrate, it is
unlikely that sweet receptors signal the brain in the study by Carter et al. Rats may have
receptors for polycose, a partially hydrolyzed, non-sweet carbohydrate similar to
maltodextrin (Nissenbaum & Scalafani, 1987). Furthermore, polysaccharide receptors are
known to exist in several species including rats, gerbils, mice, and hamsters (Feigin et al.,
1987). It is currently unknown if such receptors exist in humans.
According to Katz et al. (2000), humans possess what is called the “mouth feel
phenomenon.” Humans are able to differentiate viscosity and feel of foodstuffs in the mouth,
stimulating cortical taste neurons and orbitofrontal cortex neurons. Studies using
neuroimaging have demonstrated that oral ingestion of glucose stimulates the primary taste
cortex and the putative secondary taste cortex of the orbitofrontal cortex. The primary taste
cortex and orbitofrontal cortex are thought to have projections to the reward and behavior
centers of the brain – the dorsolateral prefrontal cortex, the anterior cingulated cortex, and
ventral striatum (Rolls, 2007). These brain regions, when activated, may influence emotion
and behavior, thereby potentially influencing exercise performance (Kringelbach, 2004).
Carter and colleagues (2004b) therefore suggested that non-sweet receptors may exist in the
human mouth capable of initiating a centrally mediated effect on exercise performance.
Chambers et al., (2009) recruited competitive or recreational cyclists (six males, two
females) to determine the effects of oral glucose and oral maltodextrin on exercise
performance. Participants were given 6.4% glucose, maltodextrin, or placebo at the start of
45
exercise and each 12.5% of time trial (equal to one hour at 75% maximum power output)
completion and instructed to rinse the mouth for approximately ten seconds and spit the
solution into a bowl held by an investigator. Subjective appraisal of sweetness and viscosity
were assessed by visual analog scales with an anchor point at 0mm labeled “Nil” and 100mm
labeled “Extreme.” Participants rated no difference between glucose and placebo solutions
for sweetness, but did rate the glucose solution as more viscous. Participants completed the
same amount of work during the performance trial in less time during the glucose trial than
the placebo trial (60.4 ± 3.7 vs. 61.6 ± 3.8 min, respectively). Average power output was
higher during the glucose trial than the placebo trial. Treatment had no effect on ratings of
perceived exertion or heart rate. For the maltodextrin study, no differences in ratings of
sweetness and viscosity were found between the maltodextrin and placebo solutions and
subjects were able to differentiate between solutions. Participants completed the performance
trial faster when receiving the maltodextrin solution than when receiving the placebo solution
(62.6 ± 4.7 vs. 64.6 ± 4.9 min, respectively). Treatment had no effect on ratings of perceived
exertion or heart rate during the performance trial.
During cycling time trials, there is a trend for decreasing power output during the trial
until a final sprint to the finish, during which power output is higher. Accordingly,
participants alter their power outputs to maintain a relatively consistent perception of
exertion (Cole et al. 1996). Chambers et al. (2009) suggested that oral exposure to
carbohydrate induces a central response that allows participants to increase power output by
reducing perception of a given workload. The authors suggest that improvements in exercise
performance were independent of the sweetness of treatments.
46
Chambers et al., (2009) also examined the regions of the brain activated by in
response to glucose, maltodextrin, and the artificial non-caloric sweetener, saccharin entering
the mouth. To assess activated brain regions, they used functional magnetic resonance
imaging to determine the blood oxygenation level-dependent responses to different solutions
in the mouth. A plastic tube connected to a reservoir via a syringe and a two-way tap,
allowed 2.5 ml of solution to be delivered to the participants. After a ten-second rest period,
2.5 ml of solution was delivered to the mouth. Participants were instructed to make one
tongue movement to distribute the solution in the oral cavity, and swallow the solution after
ten seconds of solution being in the oral cavity. After an 18-second rest period, control
solution (25 mmol KCL and 2.5 mmol NaHCO3 in distilled water) was delivered. The
participants were instructed to swallow (within two seconds) again after ten seconds had
elapsed. Each of the 12 trials lasted 52 seconds. Oral exposure to glucose resulted in
activation of insula/frontal operculum (the putative human primary taste cortex), the left and
right areas of the dorsolateral prefrontal cortex (a region of the right caudate that forms part
of the striatum; believed to be involved in preparation and selection of cognitive responses
induced by taste stimuli), and an anterior area of the cingulated cortex. Oral exposure to
saccharin evoked activation of the insula/frontal operculum and the left dorsolateral
prefrontal cortex. In contrast to oral glucose exposure, oral saccharin exposure did not result
in activation of the anterior cingulate cortex or striatum. A conjunction overlay mask was
created to examine cortical regions that were activated by both caloric (glucose) and noncaloric sweetened (saccharin) solutions. An area of the right insula/frontal operculum and a
region of the left dorsolateral prefrontal cortex were responsive to both glucose and saccharin
solutions. Oral exposure to glucose in the second study resulted in activation of the
47
insula/frontal operculum, an area of the medial orbitofrontal cortex that extended into the
bordering anterior cingulated cortex, the dorsolateral prefrontal cortex, a more dorsal region
of the anterior cingulate cortex, and the left and right caudate. Oral exposure to maltodextrin
resulted in activation of the insula/frontal operculum, medial orbitofrontal cortex that
extended into an adjoining rostral part of the anterior cingulate cortex, the dorsolateral
prefrontal cortex, and the right caudate. Oral exposure to maltodextrin did not activate the
dorsal regions of the anterior cingulate cortex, unlike oral exposure to glucose. A conjunction
overlay mask showed that the right insula/frontal operculum and a small area in the left
frontal operculum, the medial orbitofrontal cortex, the dorsolateral prefrontal cortex, and the
right caudate and rostral anterior cingulate cortex were activated by both glucose and
maltodextrin taste.
Oral glucose exposure activated regions of the brain that were unaffected by oral
saccharin exposure, including the anterior cingulate cortex and the right caudate, that forms
part of the striatum, regions believed to influence the emotional and behavioral responses to
rewarding food stimuli (Berridge & Robinson, 1998; Kelley et al., 2002; Rolls, 2007). This
supports the idea that sweetness is not a requisite for activation of reward centers of the
brain. De Araujo & Rolls (2004) observed that a rostral portion of the anterior cingulate
cortex is activated in response to oral fat, suggesting these regions of the brain are stimulated
in response to the energy content of food. Chambers et al. (2009) suggested that perhaps the
caloric content of sugars may be responsible for such activation because similar cortical
activation (primary taste cortex and a medial region of the orbitofrontal cortex, the putative
secondary taste cortex) was produced by oral exposure to glucose and maltodextrin despite
48
differences in solutions sweetness. The orbitofrontal cortex is considered to be the hedonic
value region of the brain stimulated in response to an oral stimulus.
This study showed that a non-sweet carbohydrate and glucose in the mouth produced
similar cortical activation. This finding suggest that unidentified oral receptors may exist that
respond to the caloric property of carbohydrate, independent of carbohydrate sweetness
properties. The authors corroborate the findings of Carter et al. (2004b) and concluded that
presentation of carbohydrate to the oral cavity improves exercise performance during a onehour cycle time trial. Both studies examined the brain regions activated by oral glucose
exposure and some small differences were found. In the second study, glucose activated the
orbitofrontal cortex and the adjoining rostral part of the anterior cingulate cortex. These areas
were not activated by oral glucose exposure in the first study.
According to Berridge and Robinson (1998), the dopaminergic system of the ventral
striatum is involved in arousal, motivation, and motor behavior. During exercise, much
afferent information from muscles, joints, lungs, skin, and core body temperature receptors is
sent to the brain, which may be perceived as unpleasant, leading to an inhibition of motor
output, leading to central fatigue. The “Central Governor Model” of fatigue suggests that
during physical activity, people likely modify their level of exertion to maintain an
acceptable comfort level (Noakes, 2000; Lambert et al., 2005). Whereas the exact neural
pathways are not defined in such central fatigue, if reward centers are inhibited during
prolonged exercise, it is possible that presentation of foods to the oral cavity may attenuate
inhibition of these regions, thereby improving exercise performance.
The major limitations of the studies by Chambers et al. (2009) are that functional
imaging of the brain was not performed in exercise, but at rest. Perhaps the reward centers of
49
the brain activated while swilling the mouth with a carbohydrate solution at rest are not
stimulated during exercise. Additionally, the mouth rinse solutions used in the functional
imaging study had about three times the carbohydrate concentration as the solutions used in
the exercise performance study. Therefore, it is difficult state that the mouth rinse solutions
in the exercise performance study would stimulate behavior centers of the brain.
Studies have shown that runners feel better and report lower ratings of perceived
exertion when ingesting carbohydrates during exercise (Backhouse et al., 2007; Backhouse et
al., 2005). However, Whitham and McKinney (2007) showed that simply tasting a
carbohydrate solution by rinsing the mouth without swallowing did not alter ratings of
perceived exertion during a 45-minute running time trial. A major limitation of the study by
Whitham and McKinney is that running speed could not be easily adjusted; a researcher or
participant had to manually adjust treadmill speed. Therefore, Rollo et al. (2008) tested the
effects of rinsing the mouth with a carbohydrate solution (6% carbohydrate) on self-selected
running speeds in endurance-trained runners utilizing an automated treadmill that allows free
and spontaneous adjustments to running speed without manual input. The running protocol
was a ten-minute warm-up at 60% VO2 max followed by a 30-minute trial in which the
participants were asked to maintain a running speed equal to a “hard pace” or an RPE of 15.
The treadmill had a 1% grade for the entire 40 minutes. Runners were free to adjust their
running speed using the automated system. Participants were given a 25 ml bolus of
treatment every five minutes during the 30-minute run with which they rinsed their mouths
for five seconds and expectorated. Feeling Scale (FS), Felt Arousal Scale (FAS), and
Gastrointestinal comfort (GI) scales were administered every five minutes during the 30minute run. No differences were found in heart rate, body weight loss, rating of perceived
50
activation (FAS), and gastrointestinal comfort during the 30-minute run between rinsing the
mouth with 6% carbohydrate solution and rinsing with placebo. At the start of the run,
feeling score was higher in the carbohydrate trial, but no differences were found for the
remainder of the run. Self-selected speed was faster in the first five minutes of the
carbohydrate mouth rinse trial than the placebo mouth rinse trial. Additionally, participants
ran farther during the carbohydrate mouth rinse trial than the placebo trial (6584 ± 520 vs.
6469 ± 515 m, respectively). Interestingly, rinsing the mouth with carbohydrate resulted in
significantly greater estimated energy expenditure than rinsing with placebo (493 ± 54 vs.
485 ± 55 kcal, respectively).
Rinsing the mouth with carbohydrates may lower perception of exertion at a given
exercise intensity. The participants in the Carter et al. (2004b) study had greater average
power output with no increase in ratings of perceived exertion when rinsing with a
carbohydrate solution, compared to placebo. Similarly, participants in the Rollo et al. (2008)
study were able to run faster while maintaining a rating of perceived exertion of 15 or “Hard”
when rinsing with a carbohydrate solution than placebo. Rollo et al. (2008) suggested that
carbohydrate presentation to the mouth may result in a “feel good” effect. Furthermore,
recognition of carbohydrates in the mouth might relay signals of incoming energy to the
brain, resulting in increased motor behavior or exercise performance. Additionally,
recognition of carbohydrates in the mouth may result in a transient increase in psychological
response towards exercise. However, when the brain receives feedback that no additional
energy is available, both motor behavior and psychological feelings may be adjusted
accordingly. Therefore, it may be important to rinse the mouth at given intervals. The timecourse of such responses has yet to be elucidated.
51
Pottier et al. (2008), found that rinsing the mouth with a commercially available
carbohydrate-electrolyte solution resulted in a significantly shorter time to complete a time
trial equal to one hour of pedaling at participants’ preferred pedaling cadence than rinsing
with placebo. Interestingly, ingestion of the carbohydrate-electrolyte solution did not affect
time trial performance. Pottier et al. (2008) found that participants had higher mean power
output during the self-paced cycling time trial, resulting in less time to complete a given
amount of work, higher blood lactate, and no difference in heart rate or ratings of perceived
exertion when participants rinsed the mouth with a carbohydrate-electrolyte solution,
compared to a placebo. Furthermore, rinsing of a carbohydrate-electrolyte solution allows
participants to exercise at a higher power output for a given subjective evaluation of exertion.
Rinsing the mouth with the carbohydrate-electrolyte solution resulted in higher blood lactate
concentrations than rinsing with placebo. Ingestion of the carbohydrate-electrolyte solution
resulted in significantly greater blood glucose than ingestion of placebo. There was no
difference in blood glucose between rinsing the mouth with carbohydrate-electrolyte solution
or placebo. A trend for higher blood glucose (P = 0.06) was observed for intake mode; blood
glucose tended to be higher when the treatments were ingested, compared to rinsed.
The carbohydrate-electrolyte solution in the study by Pottier et al. (2008) was
comprised of a mixture mono- and disaccharide carbohydrates, suggesting that both simple
and complex carbohydrates in the oral cavity result in improved exercise performance. The
authors hypothesize that presentation of carbohydrates to the mouth may attenuate afferent
signals of fatigue from the contracting muscle, allowing participants to produce more power
at a given level of perceived exertion. The exact mechanism is yet to be elucidated, but the
authors suggest that afferent signals from carbohydrate receptors in the oral cavity may
52
attenuate afferent signals from the muscle to the brain. During fatiguing exercise, several
neuroendocrine changes occur in the brain. Changes in enkephalinergic, dopaminergic, and
serotonergic systems occur during fatiguing exercise (Hoffman et al., 1990; Bailey et al.,
1993; Persson et al., 1993; van Hall et al., 1995). These neuroendocrine changes likely
influence motivation, and pain and its tolerance, according to Gandevia (1998), thereby
potentially influencing central fatigue. Because they observed improved performance during
the rinsing of carbohydrates trial and not the ingestion of carbohydrates trial, Pottier et al.
(2008) suggested the shorter duration of carbohydrate spent in the oral cavity during the
ingestion trial may have decreased the ergogenic effects of carbohydrate presentation to the
oral cavity.
To test the effects of carbohydrate mouth rinse on performance in a practical applied
setting, Beelen et al. (2009) assessed mouth rinsing during a cycling time trial in the fed
state. Participants consumed a meal containing 39.5 ± 0.8 kJ/kg (67% carbohydrate, 13%
protein, 20% fat energy basis), providing 2.36 ± 0.04 g/kg carbohydrate, 2.25 hours before
the time trial. Participants rinsed the mouth with 25 ml of carbohydrate (6.4% maltodextrin)
or placebo (water) for five seconds at the start and every 12.5% of the time trial completed.
Time trial performance did not differ between carbohydrate mouth rinse and placebo mouth
rinse. Heart rate, RPE, and body mass loss did not differ between treatments. Because RPE
was unaffected by carbohydrate mouth rinse, the authors refute the hypothesis that rinsing the
oral cavity with carbohydrate stimulates pleasure and reward centers of the brain. The
authors suggest that putative glucose receptors in the mouth may be more important or
influential during states of glycogen depletion and less important during states of adequate
53
glycogen stores, hence the lack of effect of benefit of carbohydrate mouth rinsing observed in
the present study.
Cephalic Phase of Insulin Release
The cephalic phase of insulin release (CPIR) has been demonstrated in animals and
humans. The β-cells of the pancreas will secrete insulin before the increase in plasma
glucose. The cephalic phase of insulin secretion is a parasympathetic reflex caused by the
sight, smell, and taste of food and is of cephalic origin. The response to taste also involves
afferent signals to the central nervous system and actions that prepare the body for caloric
consumption. Plasma insulin concentrations typically rise within two minutes, peak at four
minutes, and return to baseline within ten minutes after oral stimulation (Teff & Engelman,
1996; Teff et al., 1991; Teff et al., 1993). Placing sour (acetic acid, 0.1M), salty (NaCl, 0.5
M), bitter (quinine hydrochloride, 0.01 M), and umami (sodium glutamate, 0.2 M) tasting
treatments on the tongues of rats for 45 s did not affect plasma glucose or insulin
concentrations (Tonosaki et al. 2007). Tonosaki et al. (2007) also observed that sucrose, a
sweet, caloric carbohydrate increased plasma insulin concentrations before the rise in plasma
glucose (three vs. five min-post presentation, respectively). Saccharin, a sweet, non-caloric
sugar substitute, increased plasma insulin concentrations without affecting plasma glucose
levels. Finally, starch, a caloric, non-sweet carbohydrate, had no effect on plasma insulin or
glucose concentrations. The findings of Tonosaki et al. (2007) suggest that stimulating the
oral cavity with sweet, caloric-containing carbohydrates stimulates the CPIR in rats before an
increase in plasma glucose; stimulation with sweet, non-caloric substances stimulates the
CPIR without affecting plasma glucose concentrations; stimulation with non-sweet, caloriccontaining substances does not stimulate the CPIR or affect plasma glucose concentrations.
54
Just et al. (2008) examined the effects of rinsing the mouth for 45 s and spitting out solutions
on plasma glucose and insulin in humans. They found that intra-oral stimulation with sour,
salty, bitter, and umami taste qualities did not elicit the CPIR. They also observed that intraoral stimulation of both sucrose and saccharin (sweet substances) resulted in a transient
increase in plasma insulin five min after stimulation, without affecting plasma glucose
concentrations. The group also observed an increase in plasma insulin after rinsing the mouth
with distilled water and starch. However, they posit the results were greatly influenced by
outliers, whereas the increase in insulin after rinsing with sucrose and saccharin was
observed in all participants. Robertson et al. (2001), found that 15 minutes of chewing
nutrients without swallowing resulted in an increase in plasma insulin of that peaked at 250%
of baseline concentration. It is possible that rinsing the mouth with a carbohydrate solution
increases insulin secretion and subsequent glucose uptake into muscle cells for oxidation,
thereby enhancing time trail performance. However, the effects of rinsing the mouth with
carbohydrates on insulin secretion during exercise have yet to be studied.
Mouth Rinse Summary
Carbohydrate (CHO) ingestion during prolonged exercise bouts may improve
performance when glycogen stores become depleted. However, during shorter-term bouts,
the mechanism for improvements observed in response to CHO ingestion is not known.
There may be central or nonmetabolic factors involved. Glucose infusion (one g/min) did not
improve performance in a 40 km time trial bout (Carter et al., 2004a). The group suggested
that oral carbohydrate feeding may exert its effects through a central mechanism, improving
motor drive or motivations, mediated by receptors in the mouth or gastrointestinal (GI) tract
To test the hypothesis that receptors in the mouth may stimulate a central drive mechanism,
55
Carter et al. (2004b) had participants rinse the mouth with either a carbohydrate solution (6.4
% maltodextrin). Time to complete the performance trial was significantly less (2.9%) when
participants rinsed with carbohydrate compared to water. Carter and colleagues (2004b)
therefore suggested that non-sweet receptors may exist in the human mouth capable of
initiating a centrally mediated effect on exercise performance. Chambers et al. (2009)
showed that a non-sweet carbohydrate (maltodextrin) and glucose in the mouth produced
similar cortical activation at rest and both improved exercise performance. These findings
suggest that unidentified oral receptors may exist that respond to the caloric property of
carbohydrate, independent of carbohydrate sweetness properties. These functional imaging
studies of the brain examined resting participants. Perhaps the brain reward centers activated
while swilling the mouth with a carbohydrate solution at rest are not stimulated during
exercise. Additionally, the mouth rinse solutions used in the functional imaging study had
~three times the carbohydrate concentration as the solutions used in the exercise performance
study. The cephalic phase of insulin release (CPIR) has been observed in animals and
humans. The β-cells of the pancreas secrete insulin before an increase in plasma glucose.
Oral cavity stimulation with sweet, non-caloric substances stimulates CPIR without affecting
plasma glucose concentrations. Finally, oral cavity stimulation with non-sweet, caloriccontaining substances does not stimulate the CPIR or affect plasma glucose concentrations
(Tonosaki et al., 2007). Taken together, these results suggest that mouth rinsing with both
sweet and non-sweet solutions may improve performance owing to activation of brain centers
responsible for motor output and drive. Additionally, enhancement of exercise performance
from mouth rinsing with sweet solutions may involve insulin secretion.
56
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CHAPTER 3. MOUTH RINSING WITH BRANCHED-CHAIN
AMINO ACIDS AND CARBOHYDRATES ON STEADY-STATE
METABOLISM, EXERCISE PERFORMANCE, AND THE ENDOCRINE
RESPONSES TO EXERCISE
A paper to be submitted to the Journal of Strength and Conditioning Research
Zebblin M. Sullivan1, Walter H. Hsu2, and Douglas S. King1
1
Department of Kinesiology
2
Department of Biomedical Sciences
Iowa State University,
Ames, IA 50011, USA
Abstract
Previous research suggests that mouth rinsing carbohydrates (CHO) improves
exercise performance. The mechanisms for the improvement are not yet known, but may
involve maintenance of insulin concentrations during exercise or attenuation of centrallymediated fatigue from putative, caloric-responsive receptors in the oral cavity. The purpose
of this study was to determine the effects of mouth rinsing branched-chain amino acids
(BCAA) or CHO on cycle time trial performance, blood markers of central fatigue
(prolactin), plasma insulin, glucose, and lactate concentrations, substrate oxidation during
steady-state exercise, and the subjective exercise experience. Ten healthy, endurance-trained
adult males (23.5 ± 3.8 y, 74.0 ± 9.0 kg, 180.5 ± 5.4 cm, 57.9 ± 6.9 ml/kg/min VO2 peak)
volunteered for the study. Participants exercised at 65% Wattmax for 30 min then completed a
time trial equivalent to 30 min cycling at 75% Wattmax. Participants rinsed the mouth with
6% glucose (CHO), 6% branched-chain amino acids (BCAA), or water (PLA) for 10 s at the
start and after every 10 min during the steady-state exercise and cycle time trial. Treatment
did not affect heart rate, ratings of perceived exertion, affect, arousal, plasma glucose and
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lactate concentrations, and substrate oxidation during steady-state exercise and a cycle time
trial. Furthermore, mouth rinsing BCAA or CHO did not affect plasma insulin or prolactin
concentrations during steady-state exercise or a cycle time trial. Finally, mouth rinsing
BCAA or CHO did not affect cycle time trial performance after steady-state exercise. In
conclusion, mouth rinsing BCAA or CHO does not improve cycle time trial lasting
approximately 30 min when performed after 30 min steady-state exercise.
INTRODUCTION
Attenuation of fatigue during training and competition is highly important for athletic
endeavors. Two broad types of fatigue exist – central and peripheral. Peripheral fatigue is
caused by factors outside the central nervous system, primarily in the muscle. Causes of
peripheral fatigue include depletion of phosphocreatine, depletion of glycogen, accumulation
of lactate, and malfunction of the neuromuscular transmission (3). Central fatigue refers to
fatigue originating in the central nervous system and may involve: 1) an increase in key
muscle compounds during physical activity, including H+, K+, bradykinin, phosphate, and
prostaglandins that could transmit information from fatiguing muscle to the brain; 2) a
decrease in the blood and brain glucose levels; and 3) an increase in the concentration of
tryptophan (Trp) in the blood and hence the neurotransmitter 5-hydroxytryptamine (5-HT) in
the brain (24). Plasma tryptophan is the amino acid precursor in the synthesis of 5-HT
(serotonin) in the brain. Tryptophan crosses the blood-brain barrier via the L-system, the
Large Neutral Amino Acids (LNAA) transporter (6). Because branched-chain amino acids
(leucine, isoleucine, valine; BCAA) are approximately 75% of the LNAA and are transported
via the same transport system (17), they may compete with free tryptophan for brain uptake.
Increasing the ratio of free tryptophan to BCAA (fTrp:BCAA) increases the amount of free
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tryptophan that can cross the blood-brain barrier and is thought to increase the synthesis and
release of 5-HT in the brain, causing central fatigue, which is associated with lethargy,
fatigue, and decreased exercise performance (15). The Central Fatigue Theory posits that
BCAA supplementation may decrease sensations of fatigue and lethargy and limit central
fatigue by competing with free tryptophan for brain uptake, possibly improving exercise
performance (7). Exercise decreases plasma BCAA and increases free tryptophan
concentrations (14). Plasma BCAA concentrations increase in response to BCAA ingestion
in a dose-dependent manner, but may return to baseline after one h of ingestion of less than
one g BCAA. Branched-chain amino acids supplementation has been shown to decrease
perceived exertion and increase endurance performance (7, 8) and reduce brain serotonin
synthesis (19). Others have found that BCAA administration 70-90 min before short-term
exercise does not impact physical performance (34, 35).
Carbohydrate (CHO) ingestion improves exercise performance during prolonged
exercise bouts (> 2 h) in which glycogen depletion may occur (20). However, the effects of
CHO ingestion on exercise performance in shorter-term bouts are unclear. Carbohydrate
ingestion before and during short-term and intense exercise sessions has been shown to delay
fatigue and improve exercise performance (5, 13). However, results are equivocal (25). The
mechanism underlying reported performance enhancement is not well understood. Because
only 5 to 15g of exogenous CHO are oxidized in the first h of exercise, it is unlikely that
improved exercise performance after CHO ingestion is due to enhanced CHO oxidation rates.
There may be “nonmetabolic” or central mechanisms involved in increased exercise
performance from CHO ingestion. Carter et al. (10) found that whereas intravenous glucose
infusion (one g/min) resulted in higher circulating glucose, infusion did not improve exercise
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performance. The group suggested that oral CHO feeding may exert its effects through a
central mechanism, improving motor drive or motivations, mediated by receptors in the
mouth or gastrointestinal (GI) tract. They found that simply rinsing the mouth with CHO,
without swallowing, improved exercise performance (11). Because potential stimulation
from GI tract receptors was eliminated, Carter and colleagues (11) suggested that non-sweet
receptors may exist in the human mouth, capable of initiating a centrally mediated effect on
exercise performance. Chambers et al. (12) suggested that unidentified oral receptors may
exist that respond to the caloric property of carbohydrate, regardless of sweetness and the
caloric content of solution mediates brain activation, which may in turn influence emotion,
behavior, and potentially, exercise performance. Gant et al. (18) posited that unidentified
class of receptors may exist that encode caloric information, influencing descending motor
activity. The positive effects of mouth rinsing with CHO on exercise pervious have been
supported by several subsequent studies involving fasted participants (12, 26, 27, 28), but not
those in a fed state (4).
Mouth rinsing with CHO has also been hypothesized to maintain blood insulin
concentrations during exercise, which may enhance performance by stimulating glucose
uptake into cells (11). The cephalic phase of insulin release (CPIR), a parasympathetic reflex
caused by the sight, smell, and taste of food, has been observed in animals and humans. The
β-cells of the pancreas secrete insulin before an increase in plasma glucose. Recent work has
shown that sweet substances (caloric and non-caloric alike) presented to the mouth, but not
ingested, stimulate the CPIR before a rise in plasma glucose concentrations (23, 33).
This study will determine if mouth rinsing with other nutrients (BCAA) enhances
exercise performance as measured by a cycle time trial. By using isocaloric solutions from
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different nutrient sources (CHO and BCAA), this study will answer the question if the
previously observed improvements in exercise performance from CHO mouth rinsing with
were due to carbohydrates per se or the presentation of energy to the oral cavity.
Furthermore, because BCAA are bitter and the CHO in the study is sweet, the study will
determine if sweetness is a requisite property of mouth rinse solutions that improve exercise
performance. Additionally, the effects of mouth rinsing with CHO or BCAA on the
concentration of blood glucose during exercise, the steady-state metabolic response to
exercise, and the pancreatic secretion of insulin during exercise are not known. Another
major aim of the study is to determine the effects of rinsing the mouth with CHO or BCAA
during exercise on markers of central fatigue. Plasma prolactin has been shown to be a
surrogate index of brain serotonin levels (37) and will used as the blood marker of central
fatigue.
METHODS
Experimental Approach to the Problem
All exercise tests were performed on an electronically braked cycle ergometer (Lode
Excalibur, Groningen, The Netherlands). During the first visit to the laboratory, participants
performed a graded exercise test to exhaustion to determine maximal aerobic capacity (VO2
peak). Visit two was a familiarization session to familiarize participants with the steady-state
metabolic response and cycle time trial procedures, subjective scales, and ensure they could
complete the required exercise. On the experimental days, participants cycled for 30 min at a
steady-state workload and were then instructed to perform a given amount of work as quickly
as possible. During the experimental trials, participants were given either a 6% glucose
solution (CHO), 6% branched-chain amino acids solution (BCAA; Leucine:Isoleucine:Valine
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= 2:1:1), or water (PLA) to rinse around the mouth for ten s before and every ten min during
exercise. Experimental trials were performed in a randomized, counter-balanced, singleblind, placebo-controlled, within-subjects crossover design. Trials were separated by at least
seven d.
Subjects
Ten healthy, endurance-trained adult males (23.5 ± 3.8 y, 74.0 ± 9.0 kg, 180.5 ± 5.4 cm,
57.9 ± 6.9 ml/kg/min VO2 peak) volunteered for the study. Training criteria included people
currently engaged in endurance exercise for 30-60 min at least three times per week at 6080% heart rate reserve and able to exercise for approximately 60 min at 75% Wmax on a cycle
ergometer. Participants were required to have a VO2 peak ≥ 50 ml/kg/min. People with a
serious illness or disease such as hypertension, kidney or liver disease were excluded from
the study. Participants were fully informed on the nature and possible risks of the
experimental procedures before their written consents were obtained. The study was
approved by the Iowa State University Human Subjects Institution Review Board.
Procedures
Maximal Aerobic Capacity
During the graded exercise test to exhaustion, participants cycled on a Lode Excalibur
cycle ergometer. Power output increased 50 watts every two min until the participant reached
volitional exhaustion. Ventilation, oxygen uptake (VO2), carbon dioxide production (VCO2),
heart rate (HR; Polar, Finland), and ratings of perceived exertion (RPE; 9) were recorded
continuously. Expired respiratory gases were collected and analyzed using a Max II
Physiodyne metabolic cart (Physiodyne Instrument Corp, Quogue, NY) calibrated according
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to manufacturer’s instructions. Seat position, handlebar height and orientation were recorded
and replicated in each subsequent visit.
Steady-State Metabolism and Time Trials
Participants entered the laboratory in the morning after an overnight fast, having
abstained from caffeine, alcohol, tobacco, and exercise in the previous 24 h. Each participant
performed the three experimental trials at the same time of day. On arrival to the laboratory,
participants were weighed and fitted with a HR monitor (Polar, Finland). A polyethylene
catheter was inserted into an antecubital vein for subsequent blood sampling. To prevent
clotting, 0.9% sodium chloride injection (3 ml) was added to the vein. After a five min
warm-up at 40% Wmax, participants cycled for 30 min at 65% Wmax to determine the effects
of mouth rinsing with CHO and BCAA on steady-state metabolism and rate of oxygen
consumption (VO2). Expired gases were collected the last two min of each ten min interval
and analyzed for VO2 and the respiratory exchange ratio (RER). Participants then performed
a cycle time trial in which they were instructed to perform a set amount of work in the
shortest time possible. Total work performed was calculated according to a modification of
the Jeukendrup et al. (21) equation:
Total amount of work = 0.75 x Wmax x 1,800
where Wmax is the maximal workload capacity determined at visit one and 1,800 is the
duration in s (equivalent to 30 min). This test has been reported to be highly reproducible
(21). The ergometer was set in the linear mode according to the formula W = L x (RPM)2 in
which RPM is the pedaling rate and L is a linear factor. The linear mode was set so that 75%
Wmax was obtained when the participants pedaled at their preferred cadence determined
during the maximal aerobic capacity test. If the participants exercised at this pedaling rate,
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they would have completed the time trial in exactly 30 min. The ergometer was connected to
a computer that calculated and displayed the total amount of work performed. The only
information the participant received during the time trial was the work performed and the
percentage of work performed relative to the preset task. No encouragement was given to the
participants and they were kept unaware of performance-related information, such as exercise
time, heart rate, and cycling cadence. A fan was placed one meter behind the participant to
provide cooling and air circulation during the trials. Heart rate was recorded continuously
throughout the steady-state exercise and time trial using short-range telemetry (Polar,
Finland). Ratings of perceived exertion (9) were recorded at the start and every ten min
during the steady-state exercise and time trial. Expired gases were collected during the last
two min of each ten min interval and analyzed for VO2 and respiratory exchange ratio (RER)
during the time trial. During the time trial, no interaction between the participant and the
investigators occurred except for subjective scales, blood draws, and mouth rinse
administration. All people not involved in the study were excluded from the laboratory to
prevent any external disruption.
Oxidation of carbohydrate and fat was estimated based on the equations for moderate to high
intensity exercise (50-75% VO2 max) by Jeukendrup & Wallis (22):
Carbohydrate oxidation (g/min) = 4.210 * VCO2 – 2.962 * VO2 – 0.40 * n
Fat oxidation (g/min) = 1.695 * VO2 – 1.701 * VCO2 – 1.77 * n
where n represents nitrogen excretion in urine. Negligible protein oxidation was assumed.
Mouth Rinse Protocol
Each participant was given a 25 ml bolus of either 6% glucose (CHO), 6% branchedchain amino acids (BCAA), or water (PLA) at the start and every 7th min of each ten min
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interval during the steady-state exercise and cycle time trial. Participants rinsed the fluid
around the mouth for ten s before expectorating the fluid into a beaker. Each 25-ml bolus of
solution was served in a plastic syringe (Kendal monoject). Participants were kept blind to
the composition of the rinse treatments until the study was completed. Beverage
administration was randomized via a random-number generator (SPSS v. 19).
Evaluation of Treatments
Participants evaluated treatment solutions for bitterness and sweetness using 100 mm
visual analog scales with anchors at 0 mm (Nil) and 100 mm (Extreme). Bitterness and
sweetness was assessed before and after the steady-state exercise and time trial.
Ratings of Perceived Exertion
Ratings of perceived exertion (9) is a scale that ranges from 6 (Very very light) to 20
(Very very hard) and is used to determine the exercise exertion level of a participant. The
RPE scale was administered at the start and every ten min during the 30 min steady-state
exercise and time trial.
Dietary and Physical Activity Procedures
To minimize intra-participant differences in starting liver and muscle glycogen
concentrations, participants were instructed to record their diet for the three d before the first
visit and to avoid exercise in the 24 h period before each visit. The three d diet record was
copied and returned to the participant with instructions to follow the same diet before each
subsequent visit. Diet compositions were analyzed using the Nutritionist Pro software
program (Stafford, TX; Table 1).
Blood Analyses
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A blood sample (10 ml) was taken from the catheter inserted into an antecubital vein
before exercise and every ten min during the steady-state exercise and the time trial. To
prevent clotting, 0.9% sodium chloride (3 ml) was added to the vein after each blood draw.
Samples were centrifuged for ten min at 2,200 rpm using an Allegra TM 6R centrifuge.
Plasma was stored at -80°C until analysis. Plasma glucose and lactate concentrations were
determined using a Yellow Springs Glucose/Lactate AutoAnalyzer (YSI Incorporated,
Yellow Springs, OH, USA). Samples were analyzed in duplicate.
Hormone Analysis
Insulin
All assays were performed in duplicate. Standard or sample (0.1 ml) was added to 12
x 75 mm tubes. First antibody (0.1 ml; guinea pig antiserum vs. porcine insulin, 1:10,000)
was added to each tube except blank tubes. Phosphate buffer with bovine serum albumin
(0.04 M) was added to blank tubes (0.2 ml) and Z tubes (0.1 ml). Tubes were incubated
overnight at 4°C. 125I-insulin (0.1 ml; ~20,000 counts per min (cpm)/0.1 ml) was added to all
tubes. Tubes were incubated overnight at 4°C. Normal guinea pig plasma (0.1 ml; 1:80) and
second antibody (0.1 ml; donkey anti-guinea pig IgG, 1:40) were added to each tube. Tubes
were shaken and incubated at room temperature for 30 min. At least two tubes were counted
during incubation for determination of total counts. Human plasma (0.1 ml) was added to
each tube in the standard curve. Polyethylene glycol (1 ml; 12% in 0.85% NaCl) was added
to each tube. Tubes were centrifuged at 3000 rpm for 30 min at 4°C. Supernatant was
aspirated and tubes were counted for one min using a gamma counter. Standard curves for
insulin concentration determination contained blank, Z, 0.78, 1.55, 3.125, 6.25, 12.5, 25, 50,
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and 100 µU/ml. Reference standards (5, 20, and 50 µU/ml) were assayed to determined interand intra-assay variability. Radioactivity was measured using an automated gamma counter.
Prolactin
All assays were performed in duplicate. Standard or sample (0.1 ml) was added to 12
x 75 mm tubes. Antibody (0.1 ml; rabbit anti-human prolactin, 1:50,000) was added to each
tube except blank tubes. Assay buffer (0.1% BSA in 0.01 M PBS with 1 mg/ml sodium
azide, pH 7.4) was added to blank tubes (0.2 ml) and Z tubes (0.1 ml). 125I-insulin (0.1 ml;
~20,000 cpm/0.1 ml) was added to all tubes. Tubes were incubated overnight at 4 C. Normal
rabbit plasma (0.1 ml; 1:80) and second antibody (0.1 ml; goat anti-rabbit IgG, 1:20) were
added to each tube. Tubes were shaken and incubated at room temperature for 30 min.
Standard curve tubes were counted during incubation for determination of total counts.
Human plasma (0.1 ml) was added to each tube in the standard curve. Polyethylene glycol (1
ml; 12% in 0.85% NaCl) was added to each tube. Tubes were centrifuged at 3000 rpm for 30
min at 4°C. Supernatant was aspirated and tubes were counted for one min using a gamma
counter. Standard curves for prolactin concentration contained blank, Z, 0.3125, 0.625. 1.25,
2.5, 5, 10, 20, and 40 ng/ml. The reference standard (16 ng/ml) was assayed to determine
inter- and intra-assay variability.
Iodination and purification of human prolactin
A Sephadex G75 column was made. A 10-ml pipet was packed with Sephadex G75
beads (1g in 20 ml of 0.01 M PBS with 1 mg/ml BSA) in room temperature. A mark was
made on the pipet 20 cm from the bottom of the column. The pipet was filled with buffer to
the 20 cm mark and beads were dropped into the column. After beads were settled in the
bottom of the column, a valve was opened allowing buffer to flow out of the pipet.
80
Additional beads were added until 20 cm were packed into the column. Buffer (3-5 ml) was
added on top of the beads. The column was stored at 4°C overnight.
The Sephadex column was removed from refrigeration one h before iodination to
bring the column to room temperature. In a 12 x 75 mm prolactin tube (5µg/5µl), 35 µl 0.3
M PO4, 1 mCi Na 125I, and 10 µl chloramine T were added at room temperature. After two
min, a small aliquot (~1 µl) was removed and added to a mixture of 1 ml TCA (10%) and 1
ml 2.5% BSA in 0.01 M PO4. The tube was centrifuged and pellet and supernatant were
counted. Na metabisulfite (10 µl) was added to stop iodination. The mixture was transferred
to the Sephadex G75 column. Fractions (0.3 ml; n=40) were collected and 5 µl of each
fraction were counted for 30 s. The first peak detected was free 125I and second peak detected
was 125I-prolactin.
Statistical Analysis
Data were analyzed using SPSS 19.0 statistical analysis software (SPSS Inc., Chicago, IL,
USA). Missing data were estimated using the series mean method for replacing missing values. Data
were screened for normality of distribution (Kolmogorov-Smirnov test) and equality of variances
(Levene’s test for equality of variances). When appropriate, non-normal data were transformed into
ranked cases for all trials combined. Ranks were then separated by trial for analysis. Data were
analyzed using a series of two-factor analyses of variance (ANOVA) with repeated measures
(treatment x time). Greenhouse-Geisser epsilon corrections were used when the assumption of
sphericity was violated. Partial η2 was used to determine effect size (ES). Post hoc or pairwise mean
differences were examined after Bonferroni adjustments. All levels of significance were set at P<
0.05. All data are presented as means ± standard deviations.
81
RESULTS
Treatment evaluation
Participants evaluated BCAA as more bitter than CHO or PLA during the steady-state
trial (78 ± 14, 6 ± 7, 11 ± 19 mm, respectively; P < 0.001, Figure 1A) and the time trial (81 ±
14, 5 ± 6, and 12 ± 21 mm, respectively, P < 0.001, Figure 1B). Conversely, participants
evaluated CHO as sweeter than BCAA or PLA during the steady-state trial (49 ± 21, 6 ± 5,
and 6 ± 6 mm, respectively, P < 0.001, Figure 2A) and the time trial (51 ± 17, 7 ± 6, and 7 ±
6 mm, respectively, P < 0.001, Figure 2B).
Substrate Oxidation
Carbohydrate oxidation
The main effect of time for the carbohydrate oxidation rate during the steady-state
trial was significant (P < 0.001, Figure 3A). The carbohydrate oxidation rate decreased
during the steady-state trial (2.83 ± 0.48, 2.69 ± 0.49, and 2.55 ± 0.56 g/min for min 10, 20,
and 30, respectively). The main treatment effect and the treatment x time interaction were not
significant.
The main treatment and time effects and the treatment x time interaction for the
carbohydrate oxidation rate during the time trial were not significant (Figure 3B).
Fat oxidation
The main effect of time for the fat oxidation rate during the steady-state trial was
significant (P < 0.001, Figure 4A). The fat oxidation rate increased during the steady-state
trial (0.43 ± 0.21, 0.54 ± 0.21, and 0.61 ± 0.22 g/min for min 10, 20, and 30, respectively).
The main treatment effect and the treatment x time interaction were not significant.
82
The main effect of time for the fat oxidation rate during the time trial was significant
(P = 0.002, Figure 4B). The fat oxidation rate during the time trial was greater at 30 min
(0.68 ± 0.22 g/min) than 10 or 20 min (0.58 ± 0.22and 0.63 ± 0.20 g/min, respectively). The
main treatment effect and the treatment x time interaction were not significant.
Heart rate
The main effect of time for heart rate during the steady-state trial was significant (P <
0.001, Table 2). The main treatment effect and the treatment x time interaction were not
significant.
The main effect of time for heart rate during the time trial was significant (P < 0.001,
Table 3). The main treatment effect and the treatment x time interaction were not significant.
Ratings of perceived exertion
The main effect of time for ratings of perceived exertion was significant (P < 0.001,
Table 2). The main treatment effect and the treatment x time interaction were not significant.
The main effect of time for ratings of perceived exertion during the time trial was
significant (P < 0.001, Table 3). The main treatment effect and the treatment x time
interaction were not significant.
Insulin
The main effect of time for insulin during the steady-state trial was significant (P <
0.001, Figure 5A). Insulin concentrations were higher pre steady-state (7 ± 8 µU/ml) than at
min 10 (6 ± 8 µU/ml) during the steady-state trial, with resting values (6 ± 8 µU/ml) being
intermediate. Insulin concentrations were lower at min 20 and 30 (5 ± 6 and 4 ± 5 µU/ml,
respectively) than resting concentrations, with min 10 being intermediate. The main
treatment effect and the treatment x time interaction were not significant.
83
The main effect of time for insulin during the time trial was significant (P < 0.001,
Figure 5B). Insulin was higher pre time trial than min 10 (7 ± 8 and 3 ± 4 µU/ml,
respectively), which in turn was higher than min 20 and 30 and post time trial (3 ± 4, 3 ± 4,
and 2 ± 4 µU/ml, respectively). The main treatment effect and the treatment x time
interaction were not significant.
Prolactin
Prolactin concentrations were not affected by treatment or time during the steadystate trial (Figure 6A) or the time trial (Figure 6B).
Lactate
The main effect of time for lactate during the steady-state trial was significant (P <
0.001, Figure 7A). Lactate decreased from rest to pre steady-state (2.5 ± 1.5 and 1.4 ± 0.4
mmol/L, respectively). Lactate increased during the steady-state trial until 20 min, which did
not differ from 30 min (3.8 ± 1.7, 4.3 ± 2.1, and 4.5 ± 2.3 mmol/L for min 10, 20, and 30,
respectively). The main treatment effect and the treatment x time interaction were not
significant.
The main effect of time for lactate during the time trial tended to be significant (P =
0.072, Figure 7B). The main treatment effect and the treatment x time interaction were not
significant.
Glucose
The main effect of time for glucose during the steady-state trial was significant (P =
0.040, Figure 8A). Glucose was higher pre steady-state than min 10 (4.9 ± 0.5 and 4.8 ± 0.5
mmol/L, respectively), with all other time points being intermediate. The main treatment
effect and the treatment x time interaction were not significant.
84
The main effect of time for glucose during the time trial was significant (P < 0.001,
Figure 8B). Glucose was higher pre time trial (5.5 ± 0.6 mmol/L) than all other time points.
Glucose at min 20 was lower than glucose at min 30, with min 10 being intermediate (4.6 ±
0.7, 4.7 ± 0.7, and 4.5 ± 0.6 mmol/L, respectively). Glucose post time trial (5.0 ± 0.8
mmol/L) was higher than min 10, 20, and 30. The main treatment effect and the treatment x
time interaction were not significant.
Time trial performance
The main treatment effect for cycle time trial performance was not significant (Figure
9). Time required to complete the cycle time trial was 35.9 ± 3.8, 36.0 ± 3.3, and 35.2 ± 2.8
min for BCAA, CHO, and PLA, respectively.
DISCUSSION
The main findings of this study are that mouth rinsing with BCAA or CHO does not
affect heart rate, ratings of perceived exertion, plasma insulin, prolactin, glucose, and lactate
concentrations, and substrate oxidation during steady-state exercise. Additionally, mouth
rinsing with BCAA or CHO does not affect cycle time trial performance after steady-state
exercise.
We chose BCAA as a rinse treatment because it has been postulated that presentation
of energy to the mouth, regardless of solution sweetness, stimulates reward centers of the
brain and may increase motor output and drive (12). Furthermore, ingestion of BCAA has
been observed by some (7, 8) but not all (34, 35) to enhance exercise performance. We found
that mouth rinsing with BCAA had no effect on exercise performance. Several studies have
demonstrated that mouth rinsing with CHO during exercise improves exercise performance
(11, 12, 26, 27, 28). However, others have observed no benefit of mouth rinsing with CHO
85
without ingestion (29) or when participants are in a fed state (4). The present study
corroborates the findings of Rollo et al. (29) that mouth rinsing with CHO does not enhance
exercise performance. Rollo et al. (29) suggested they may have failed to observe a benefit
from mouth rinsing with a carbohydrate-electrolyte (CE) solution because participants mouth
rinsed and ingested placebo and mouth rinsed and expectorated the CE. These authors
suggested that ingestion of a fluid per se may have a beneficial effect on performance. In the
present study, no treatments were ingested; therefore direct comparison to the study by Rollo
et al. (29) is challenging.
There is no clear explanation why others have observed an increase in exercise
performance in response to mouth rinsing with CHO solutions. There may be
“nonmetabolic” or central mechanisms involved in increased exercise performance involving
CHO ingestion. Carter and colleagues (11) observed improvements in exercise performance
when participants mouth rinsed with maltodextrin and suggested that non-sweet receptors
may exist in the human mouth capable of initiating a centrally mediated effect on exercise
performance. Chambers et al. (12) corroborated the findings of Carter et al. (11) and found
that mouth rinsing with sweet (glucose) and non-sweet solutions (maltodextrin) may
enhanced exercise performance in bouts lasting ~ one h. Pottier et al. (26) found that mouth
rinsing with a commercially available CE solution resulted in less time to complete a one h
time trial than rinsing with placebo, whereas ingestion of CE did not. Pottier et al. (26)
suggested that afferent signals from CHO receptors in the oral cavity may attenuate afferent
signals from the muscle to the brain. Gant et al. (18) found that ingestion of CHO
immediately improved performance by increasing corticomotor excitability and maximal
voluntary force production. Additionally, Gant et al. (18) posited that because the CHO in
86
their study was non-sweet maltodextrin, signal transduction by receptors sensitive to energy
density, not sweetness may occur. The authors hypothesized that separate binding sites on the
sweet receptor may exist for sweetness and energy density or unidentified class of receptors
may exist that encode caloric information. The results of the current study do not support the
previous findings that rinsing the mouth non-sweet or sweet solutions improve exercise
performance.
In the study by Beelen et al. (4), mouth rinsing with CHO did not affect time trial
performance when participants were in a fed state. Because RPE were unaffected by
carbohydrate mouth rinse, the authors refute the hypothesis that rinsing with the oral cavity
with carbohydrate stimulates pleasure and reward centers of the brain. Beelen et al. (4)
suggested that putative glucose receptors in the mouth may be more important or influential
during states of glycogen depletion and less important during states of adequate glycogen
stores, hence the lack of effect of benefit of mouth rinsing with CHO in their study. In the
present study, participants exercised for approximately one h. Whereas muscle glycogen
content was not measured, it is likely that adequate stores were available for use; therefore, it
is possible that ergogenic effects of mouth rinsing with BCAA or CHO were not observed
due to adequate muscle glycogen content. Future research on the effects of rinsing the mouth
with energy-containing solutions during long-term exercise in which glycogen depletion
occurs is therefore warranted.
The cephalic phase of insulin release (CPIR) has been observed in animals and
humans. The β-cells of the pancreas secrete insulin before an increase in plasma glucose. The
CPIR is a parasympathetic reflex caused by the sight, smell, and taste of food and is of
cephalic origin. Whereas the secretion of insulin during the CPIR is relatively small, it plays
87
an important role in preparing the body for caloric consumption by preventing large increases
in blood glucose after eating (2). Taste qualities of foodstuffs may influence the CPIR.
Placing sour, salty, bitter, and umami treatments on the tongues of rats for 45 s did not affect
plasma glucose or insulin concentrations (33). However, Tonosaki et al. (33) found that
sweet, caloric-containing CHO stimulated the CPIR in rats before an increase in plasma
glucose; sweet, non-caloric substances stimulated the CPIR without affecting plasma glucose
concentrations and non-sweet, caloric-containing substances did not stimulate the CPIR or
affect plasma glucose concentrations. Just et al. (23) observed a similar effect in humans.
They found that whereas intra-oral stimulation with sour, salty, bitter, and umami taste
qualities did not elicit the CPIR, intra-oral stimulation of both sucrose and saccharin (sweet
substances) resulted in a transient increase in plasma insulin five min after stimulation. It has
been hypothesized that mouth rinsing with CHO during exercise may maintain insulin levels,
thereby enhancing glucose availability and subsequently CHO oxidation rates, enhancing
exercise performance exercise (11). To our knowledge, the present study is the first to
investigate the effects of mouth rinsing with CHO or BCAA on insulin secretion during
exercise. Whereas plasma insulin concentrations decreased during exercise, we found that
mouth rinsing with a bitter (BCAA) or sweet (CHO) solution had no effect on plasma insulin
concentrations during steady-state exercise or a cycle time trial. Our findings corroborate
those of others (23, 33) that bitter solutions (BCAA) do not stimulate the CPIR. However, it
should be noted that in the present study, plasma insulin concentrations were measured
during exercise, whereas the studies by Tonosaki et al. (33) and Just et al. (23) did not
involve exercise. Rollo et al. (27), found that plasma insulin concentrations did not change
during 60 min of rest and were unaffected by mouth rinsing with a 6.4% CE solution. Plasma
88
insulin concentrations typically rise within two min, peak at four min, and return to baseline
within ten min after oral stimulation (30, 31, 32). We obtained blood samples approximately
three min after mouth rinsing with BCAA or CHO. Therefore, it is possible that insulin
secretion was stimulated by mouth rinsing with BCAA or CHO, but we did not observe the
secretion due to sampling timing. However, any such effect of mouth rinsing with on insulin
secretion did not influence exercise performance or substrate oxidation in the present study.
In the present study, plasma insulin and glucose concentrations and the CHO and fat
oxidation rates were not influenced by mouth rinsing with BCAA or CHO. Our findings
corroborate those of Rollo et al. (29), who found that mouth rinsing with a CE solution does
not affect the CHO oxidation rate during a time trial, whereas rinsing with and ingesting CE
does increase the CHO oxidation rate during exercise. Pottier et al. (26) found no difference
in blood glucose between mouth rinsing with a CE solution and placebo. In the present study,
blood glucose concentrations during steady-state exercise or a cycle time trial were not
affected by rinsing with CHO, BCAA, or PLA. Our findings corroborate those of Rollo et al.,
(2010), who found no changes in plasma glucose concentrations from pre- to post-exercise
when participants mouth rinsed with CHO. Therefore, the improvement in exercise
performance observed in other studies likely does not involve greater availability of blood
glucose for oxidation.
The effects of mouth rinsing with CHO on ratings of perceived exertion are
equivocal. Whitham and McKinney (36) found that mouth rinsing with a carbohydrate
solution did not alter RPE during a 45 min running time trial. Rollo et al. (29) also observed
no effect of mouth rinsing with CHO on RPE. Our results corroborate these findings that
mouth rinsing with BCAA or CHO does not affect RPE during steady-state exercise or a time
89
trial. However, others have suggested that mouth rinsing with CHO may lower perception of
exertion at a given exercise intensity (11, 26, 28, 12). Pottier et al. (26) hypothesized that
presentation of CHO to the mouth may attenuate afferent signals of fatigue from the
contracting muscle, allowing participants to produce more power at a given level of
perceived exertion. Rollo et al. (28) suggested that carbohydrate presentation to the mouth
may result in a “feel good” effect. Rollo et al. (28) speculated that the brain may monitor the
physiological integrity of muscle or whole-body carbohydrate stores. Presentation of CHO to
the mouth may relay signals to the brain of an incoming energy source. This promise of
energy may increase generate a transient increase in power output or exercise performance
and heighten pleasure. However, when the brain receives feedback that no energy source
arrived, exercise behavior and affect or mood state may be adjusted accordingly. Therefore, it
may be important to rinse the mouth at given intervals. The time-course of such responses
has yet to be elucidated.
Interestingly, prolactin concentrations did not change from resting levels during
exercise, which conflicts with other findings (1, 38). However, prolactin concentrations do
not increase during exercise at intensities corresponding to a VO2 of less than 40 ml/kg/min
(38), or below the ventilatory threshold (16). Since theVO2 was ~ 40 ml/kg/min during the
time trial in the present study, the exercise intensity may have not been stressful enough to
stimulate prolactin secretion. However, because prolactin concentrations did not increase
during either the steady-state exercise or the time trial, it is possible that serotonin activity
was attenuated during exercise in the present study from mouth rinsing with fluids per se. If
serotonin activity was attenuated during exercise by mouth rinsing with BCAA or CHO,
90
these neuroendocrine responses did not influence motor output as measured by cycle time
trial performance.
In conclusion, mouth rinsing with BCAA or CHO does not affect cycle time trial
performance after steady-state exercise. Mouth rinsing with BCAA or CHO does not affect
heart rate, ratings of perceived exertion, plasma glucose and lactate concentrations, and
substrate oxidation during steady-state exercise and a cycle time trial. Markers of central
fatigue (plasma prolactin concentrations) were also not affected by mouth rinsing with
BCAA or CHO. Mouth rinsing with BCAA or CHO did not affect plasma insulin
concentrations during steady-state exercise or a cycle time trial.
PRACTICAL APPLICATIONS
The findings of this study suggest that mouth rinsing with carbohydrates or branchedchain amino acids during moderate-intensity cycling does not affect exercise performance. A
coach seeking nutritional means of improving sport and exercise performance may suggest
mouth rinsing and ingesting carbohydrates during training or competition. However, this
study provides no evidence that only mouth rinsing carbohydrates improves exercise
performance.
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94
Tables and Figures
Table 1. Analysis of three d diet records. Values are mean ± SD.
Energy, kcal
Carbohydrate, g
Protein, g
Fat, g
Day 1
2866 ± 1158
340 ± 163
131 ± 54
109 ± 58
Day 2
2854 ± 954
341 ± 143
130 ± 48
110 ± 48
Day 3
2245 ± 822
271 ± 125
102 ± 47
88 ± 35
Table 2. Heart rate (HR) and ratings of perceived exertion (RPE) during steady-state
exercise. Values are mean ± SD. Time points within a measure with different superscripts are
different (P < 0.05).
Measure/Trial
Heart rate,
bts/min
BCAA
CHO
PLA
RPE
BCAA
CHO
PLA
Rest
Steady-State (Time)
Pre
10
20
30
74 ± 16a
73 ± 8a
74 ± 14a
77 ± 12a
81 ± 15a
78 ± 14a
151 ± 10b
155 ± 15b
153 ± 20b
157 ± 17c
160 ± 14c
157 ± 18c
161 ± 18c
162 ± 16c
155 ± 15c
6 ± 0a
6 ± 0a
6 ± 0a
7 ± 1a
6 ± 1a
6 ± 1a
13 ± 2b
12 ± 2b
11 ± 4b
14 ± 2c
14 ± 2c
14 ± 2c
15 ± 2d
14 ± 1d
14 ± 3d
Table 3. Effects of mouth rinsing with BCAA and CHO on heart rate (HR) and ratings of
perceived exertion (RPE) during cycle time trial. Time points within a measure with different
superscripts are different (P< 0.05).
Time Trial (Time)
Measure/Trial
Heart Rate,
bts/min
BCAA
CHO
PLA
RPE
BCAA
CHO
PLA
Pre
10
20
30
Post
105 ± 13a
107 ± 12a
106 ± 13a
160 ± 19b
163 ± 14b
163 ± 18b
164 ± 16b
164 ± 15b
163 ± 18b
163 ± 14b
167 ± 13b
164 ± 18b
170 ± 16c
170 ± 16c
168 ± 23c
7 ± 2a
7 ± 2a
7 ± 1a
14 ± 3b
14 ± 1b
14 ± 2b
15 ± 2c
15 ± 2c
15 ± 2c
16 ± 2d
15 ± 2d
15 ± 2d
16 ± 2d
15 ± 4d
16 ± 2d
95
*
*
Figure 1A. Evaluation of BCAA, CHO, and PLA bitterness pre- and post-steady-state
exercise. Values are mean + SD. * BCAA more bitter than CHO or PLA (P < 0.001).
*
*
Figure 1B. Evaluation of BCAA, CHO, and PLA bitterness pre- and post-cycle time trial.
Values are mean + SD. * BCAA more bitter CHO or PLA (P < 0.001).
96
*
*
Figure 2A. Evaluation of BCAA, CHO, and PLA sweetness pre- and post-steady-state
exercise. Values are mean + SD. * CHO sweeter than BCAA or PLA (P < 0.001).
*
+
*
Figure 2B. Evaluation of BCAA, CHO, and PLA sweetness pre- and post-cycle time trial.
Values are mean + SD. * CHO sweeter than BCAA or PLA (P < 0.001). + Solutions
evaluated as sweeter post-time trial than pre-time trial (P < 0.05).
97
a
b
c
Figure 3A. The CHO oxidation rate in response to mouth rinsing with BCAA, CHO, and
PLA during steady-state exercise. Values are means ± SD. Time points with different
superscripts are different (P < 0.05).
Figure 3B. The CHO oxidation rate in response to mouth rinsing with BCAA, CHO, and
PLA during the cycle time trial. Values are means ± SD.
98
b
c
a
Figure 4A. The fat oxidation rate in response to mouth rinsing with BCAA, CHO, and PLA
during steady-state exercise. Values are means ± SD. Time points with different superscripts
are different (P < 0.05).
b
a
a
Figure 4B. The fat oxidation rate in response to mouth rinsing with BCAA, CHO, and PLA
during the cycle time trial. Values are means ± SD. Time points with different superscripts
are different (P < 0.05).
99
a
ab
bc
c
c
Figure 5A. Plasma insulin response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise. Values are means + SD. Time points with different superscripts are
different (P < 0.05).
a
b
c
c
c
Figure 5B. Plasma insulin response to mouth rinsing with BCAA, CHO, and PLA during the
cycle time trial. Values are means + SD. Time points with different superscripts are different
(P < 0.05).
100
Figure 6A. Plasma prolactin response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise. Values are means ± SD.
Figure 6B. Plasma prolactin response to mouth rinsing with BCAA, CHO, and PLA during
the cycle time trial. Values are means ± SD.
101
d
d
c
a
b
Figure 7A. Plasma lactate response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise. Values are means ± SD. Time points with different superscripts are
different (P < 0.05).
Figure 7B. Plasma lactate response to mouth rinsing with BCAA, CHO, and PLA during the
cycle time trial. Values are means ± SD.
102
ab
a
b
ab
ab
Figure 8A. Plasma glucose response to mouth rinsing with BCAA, CHO, and PLA during
steady-state exercise. Values are means ± SD. Time points with different superscripts are
different (P < 0.05).
Figure 8B. Plasma glucose response to mouth rinsing with BCAA, CHO, and PLA during the
cycle time trial. Values are means ± SD. Time points with different superscripts are different
(P < 0.05).
103
Figure 9. Time to complete the cycle time trial when mouth rinsing with BCAA, CHO, and
PLA. Values are means ± SD.
104
CHAPTER 4. GENERAL CONCLUSIONS
The results of this dissertation research suggest that mouth rinsing with branchedchain amino acids and carbohydrates does not influence exercise performance in cycle time
trials lasting approximately 30 min when performed after 30 min of steady-state exercise.
Whereas there are several previous studies showing that mouth rinsing with carbohydrates
improves performance, coaches and athletes should be aware that mouth rinsing with
carbohydrates and branched-chain amino acids may not improve exercise or sport
performance. However, one cannot rule out the possibility of a “placebo effect.” Athletes
who have read or heard that mouth rinsing with carbohydrates improves exercise
performance may realize positive performance benefits from mouth rinsing with
carbohydrates simply because they think expect a benefit from mouth rinsing carbohydrates.
This placebo effect has been demonstrated in a classic study of anabolic steroids (Ariel &
Saville, 1972).
This dissertation research also shows that mouth rinsing with carbohydrates and
branched-chain amino acids does not influence substrate oxidation or plasma glucose or
insulin concentrations during steady-state exercise or during a cycle time trial. Therefore,
exercise metabolism is not affected by mouth rinsing with carbohydrates or branched-chain
amino acids. Additionally, mouth rinsing with sweet (carbohydrates) and non-sweet or bitter
(branched-chain amino acids) solutions does not influence insulin secretion during exercise.
Any potential ergogenic effect of mouth rinsing with carbohydrates is therefore likely due to
nonmetabolic or central factors. We also conclude from this research that mouth rinsing with
carbohydrates and branched-chain amino acids does not affect central fatigue, as measured
by the surrogate index of serotonin – plasma prolactin concentrations. Finally, mouth rinsing
105
with carbohydrates and branched-chain amino acids does not affect the subjective exercise
experience during 30 min steady-state exercise or a cycle time trial, as measured by ratings of
perceived exertion, feeling scale, and felt activation scale.
There remains a need for additional research in the area of mouth rinsing with energycontaining solutions on exercise performance. To date, the effects of mouth rinsing with
carbohydrates on anaerobic performance are not known. Additionally, the effects of mouth
rinsing with carbohydrates on longer duration exercise (lasting more than one h) have yet to
be determined. Whereas ingestion of branched-chain amino acids may improve aerobic
exercise performance, we have demonstrated that mouth rinsing with branched-chain amino
acids does not influence aerobic exercise performance. Recent work has shown that mouth
rinsing and ingesting carbohydrates increased distance ran in one h versus only mouth rinsing
carbohydrates (Rollo et al., 2011). A study designed to determine the effects of mouth rinsing
and ingesting branched-chain amino acids is therefore warranted.
106
Original Manuscript
Nutritional Impact of Branched-Chain Amino Acids on Muscle Recovery after
Eccentric Exercise
A paper to be submitted to the Journal of Strength and Conditioning Research
Zebblin M Sullivan, Shawn M. Baier, Neil M. Johannsen, and Douglas S. King
Department of Kinesiology, Iowa State University,
Ames, IA 50011, USA
Running head: BCAA and muscle recovery
This manuscript contains material that is original and not previously published in text or on
the Internet, nor is it being considered elsewhere until a decision is made as to its
acceptability by the JSCR Editorial Review Board.
Disclosure of Funding: Otsuka Pharmaceutical Co., Ltd., Japan
Please address correspondence to:
Douglas S. King, PhD
Department of Kinesiology
248 Forker Building
Iowa State University
Ames, IA 50011-1160
Phone: 515-509-3854
Fax: 515-294-8740
E-mail: [email protected]
107
ABSTRACT
Previous research suggests that branched-chain amino acids (BCAA) supplementation
may attenuate perception of muscle soreness after exercise. The purpose of this study was to
determine the effects of acute BCAA supplementation before exercise on muscle function,
appraisal of muscle soreness, and plasma creatine phosphokinase (CPK) and lactate
dehydrogenase (LDH) activities after eccentric exercise. Forty-five middle-aged people (25
women, 20 men; 48 ± 6 y) volunteered for this randomized double-blind study. Participants
ingested either 8 g BCAA (Ile:Leu:Val = 1:2:1) or placebo (PL) and immediately performed
6 sets of 10 repetitions of eccentric knee extensions and flexions at 120% of peak concentric
torque. A 5-repetition isokinetic muscle function test of knee extensions and flexions was
performed before and 1 and 2 days after eccentric exercise. A 4-wk washout period separated
the two trials. Sub-group analysis revealed the change in mean extension power during the 5repetition test from Day 1 to Day 2 was different for BCAA and PL in women (P = 0.02,
Effect size (ES) = 0.67). The change in mean flexion power from Day 1 to Day 2 was also
different for BCAA and PL in women (P = 0.03, ES = 0.65). Ratings of muscle soreness and
plasma CPK and LDH activities were mild and not different between BCAA and PL. These
results suggest that a single dose of BCAA eliminates the loss in muscle power output
observed in women after exercise that produces mild muscle damage. BCAA
supplementation did not reduce plasma markers of muscle damage.
KEY WORDS: ergogenic aids, sport drinks, muscle damage, branched-chain amino acids
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INTRODUCTION
Branched-chain amino acids have been reported to stimulate maintenance of immune
function after endurance exercise (3), promote faster buffer capacity adaptation to training
(36), increase exercise time to exhaustion (24), increase exercise time to exhaustion in the
heat (22), increase oxygen-carrying capacity in the blood (26), improve cognition after an
endurance race (14), and increase voluntary physical activity in rats (32). Additionally,
branched-chain amino acids, particularly leucine, play an important role in protein
metabolism. Leucine promotes protein synthesis and inhibits protein degradation, thereby
potentially increasing protein accretion (5). Furthermore, BCAA supplementation before
exercise may attenuate endogenous muscle protein catabolism (20).
Exercise causes damage to the skeletal muscle fibers, which lasts for several days
(12). Damage to the sarcolemma results in marked elevations in plasma levels of muscle
enzymes such as creatine phosphokinase (CPK; 18). Chronic BCAA supplementation may
suppress the rise in serum creatine phosphokinase activity for several days after exercise (8).
Acute ingestion of BCAA before squat exercise decreased subjective evaluation of muscle
soreness for three days after exercise (31). Additionally, BCAA ingestion before and at 60
minutes of a 90-minute cycling bout decreased serum CPK levels 4-, 24-, and 48-hours after
exercise, and serum lactate dehydrogenase (LDH) levels 4 hours after exercise, compared to
placebo (11). While the exact mechanism has yet to be elucidated, BCAA supplementation
may attenuate endogenous muscle protein catabolism, muscle fiber membranes may be
enhanced, thereby decreasing membrane leakiness.
Delayed-onset muscle soreness (DOMS), which typically occurs one to five days
after exercise and accompanies muscle membrane damage, can adversely affect exercise or
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sport performance. For example, concentric elbow extension torque decreased after eccentric
exercise and was lowest one day after eccentric exercise (33). The ability of muscles to
generate force can be attenuated, thereby negatively influencing performance in the shortterm (2).
The mechanism responsible for the potential prophylactic effect of branched-chain
amino acids on muscle damage is currently unknown. However, the attenuation of exerciseinduced protein degradation and stimulation of protein synthesis by BCAA supplementation
may be involved (30). Branched-chain amino acids activate enzymes in the signaling
pathway in protein synthesis after exercise particularly stimulating phosphorylation of p70S6k
protein at Ser424/Thr421 and phosphorylation of protein p70S6k at Thr389. This signaling
cascade may enhance protein synthesis during skeletal muscle recovery after resistance
exercise (4, 13). A single BCAA supplement (2 g BCAA plus 0.5 g arginine) at minute ten of
the first of three 20-minute cycling exercise bouts at 50% maximal work intensity
significantly reduced exercise-induced skeletal muscle proteolysis, measured by
phenylalanine release from the working leg during the third exercise bout (21).
The effects of an acute BCAA supplementation on muscle function, plasma enzyme
activities, and subjective evaluation of muscle soreness are not fully understood. Therefore,
the purpose of the present study was to determine the effects of an acute BCAA
supplementation before exercise on subjective appraisal of muscle soreness, plasma creatine
phosphokinase and lactate dehydrogenase activities, muscle torque and power, and ratings of
perceived exertion.
METHODS
Experimental Approach to the Problem
110
To investigate the effects of acute BCAA supplementation before exercise on muscle
function, appraisal of muscle soreness, and plasma creatine phosphokinase and lactate
dehydrogenase activities after eccentric exercise, forty-five middle-aged people completed
this randomized, double-blind study. Participants ingested either 8 g BCAA (Ile:Leu:Val =
1:2:1) or placebo and immediately performed 6 sets of 10 repetitions of eccentric knee
extensions and flexions at 120% of peak concentric torque. A 5-repetition isokinetic muscle
function test of knee extensions and flexions was performed before and 1 and 2 days after
eccentric exercise. A 4-week washout period separated the two trials.
Subjects
An email was sent to all faculty and staff at Iowa State University requesting men and
women volunteers age 40-60 years for an exercise and nutrition study. Potential participants
were screened for height, mass, body composition, blood pressure, and heart rate prior to
enrollment in the study. Body composition was estimated by bioelectrical impedance analysis
using a Quantum X Bioelectrical Body Composition Analyzer (RJL Systems; Clinton Twp.,
MI). Potential participants also completed a short training session on a dynamometer exercise
machine (BiodexTM, Shirley, NY) performing knee extensions and flexions. Persons not
meeting any exclusion criteria (highly endurance or resistance trained, ingesting protein
supplements or heart patients) were included in the study. Participant characteristics are
provided in Table 1. Baseline plasma creatine phosphokinase and lactate dehydrogenase
values were higher in men than women (P ≤ 0.05). There was no difference between men and
women in age or body mass index (BMI). The study was approved by the Iowa State
University Human Subjects Institution Review Board. Participants gave their informed
consent before inclusion in the study.
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Procedures
Participants enrolled in this study completed two three-day trials in a randomized,
double-blind cross-over design separated by a washout period of at least four weeks. Prior to
each three-day trial, participants were asked to refrain from strenuous exercise for three days
before the trial until completion of the trial. Participants entered the lab at 06:00 or 07:30
hours after an overnight fast. Body mass, heart rate, and blood pressure were measured.
Subjective muscle soreness of the right leg (0-9; 0, no soreness; 9, unbearable soreness; 17)
was assessed. A small blood sample was taken from an antecubital vein using a sterile 21
gauge Butterfly® needle. To prevent clotting, 0.9% sodium chloride injection (3 ml) was
added to the vein. After five minutes, blood (5 ml) was collected in 16 x 100 mm Monoject*
blood collection tubes containing 15% EDTA (K3) liquid. Samples were centrifuged for ten
minutes at 2200 rpm using an Allegra TM 6R centrifuge. Red blood cells were separated from
plasma and discarded. Plasma was stored in Fisherbrand® 12 x 75 mm polypropylene culture
test tubes. Plasma for CPK analysis was frozen at -80°C until analysis.
Participants were fed a small meal (200 g jelly-like food; 100 kcal) (Otsuka
Pharmaceutical) to break fasts. Thirty minutes post-ingestion, participants performed a fiverepetition isokinetic strength and power test of right leg concentric knee extensions and
flexions at 1.05 rad/s (60°/s) using a dynamometer (BiodexTM, Shirley, NY). Peak torque and
average power were recorded. Immediately after the strength test, participants were given a
500 ml drink containing either 8 g branched-chain amino acids (BCAA)
(Isoleucine:Leucine:Valine = 1:2:1) or taste-, volume-, and color-matched placebo (PL).
Participants were blinded to treatment to prevent any potential placebo effects on scores of
subjective scales. Participants then performed six sets of ten repetitions of isokinetic
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eccentric right knee extensions and flexions at 1.05 rad/s (60°/s). One-minute rest intervals
were given between sets. Participants were verbally encouraged to maintain a peak torque
equal to or greater than 120% of peak concentric torque. Participants were also allowed to
view the computer monitor to observe their torque during the eccentric exercise. Ratings of
perceived exertion (RPE; 6) were measured after the first, third, and sixth sets. After
eccentric exercise, subjective muscle soreness was assessed; heart rate and blood pressure
were measured. A second small blood sample (5 ml) was taken. Heart rate and blood
pressure were measured fifteen minutes post-exercise. Participants were requested to refrain
from eating or drinking (except water) for two hours after leaving the lab to prevent
confounding results from additional nutrients.
Participants entered the lab 24 and 48 hours later after an overnight fast. Body mass,
heart rate, and blood pressure were measured. Subjective muscle soreness of the right leg was
assessed. A small blood sample (5 ml) was taken. A five-repetition isokinetic strength and
power test was performed. Ratings of perceived exertion were measured. After the strength
and power test, subjective muscle soreness was assessed; heart rate and blood pressure were
measured.
Plasma CPK enzyme activities were analyzed in duplicate after all samples were
collected. Plasma LDH enzyme activities were analyzed in duplicate within 48 hours. Plasma
lactate dehydrogenase and creatine phosphokinase enzyme activities were analyzed using
Pointe Scientific, Inc. (Canton, MI) reagent sets, according to manufacturer’s instructions (9;
14).
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At least four weeks after the first trial, participants completed a second trial. The
second trial was identical to the first with the exception of participants receiving the other
treatment.
Statistical Analyses
Data were analyzed using SPSS 15.0 statistical analysis software. Sample size was
determined based on power analysis using a power to observe differences set at 0.8. Missing
data were replaced with the mean of the corresponding variable. This was accomplished by
the series mean method for replacing missing values. Data were screened for normality of
distribution and equality of variances. When appropriate, non-normal data were transformed
into ranked cases from smallest to highest for all trials combined. Ranks were then separated
by trial number for analysis. Data were analyzed using ANOVA with repeated measures on
the second and third factor (gender x treatment x time). Partial η2 was used to determine
effect size (ES). Post hoc or pairwise mean differences were examined after Bonferroni
adjustments. Differences between men and women were tested at baseline by independent ttests. Change scores were analyzed by independent t-tests (trial). Effect sizes of change
scores were calculated using pooled standard deviations to determine meaningfulness. Level
of significance was set at P < 0.05. An a priori hypothesis was peak torque and average
power during five-repetition strength tests would be highly correlated. Pearson r for peak
torque and average power for day one, two and three was 0.965, 0.979 and 0.968,
respectively. All correlations were significant (P < 0.01). Therefore, peak torque will be
omitted from analysis and discussion. All data are presented as means ± standard errors.
RESULTS
Mean knee extension power
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Men had greater mean knee extension power output than women (P < 0.001, partial
η2 = 0.578, Figure 1). The main effects for treatment and time were not significant. The
treatment × time interaction tended to be significant (P = 0.107, partial η2 = 0.026). Because
a borderline quadratic effect was observed for the interaction of time and treatment (P =
0.085, partial η2 = 0.034), change scores were analyzed separately for men and women. Subgroup analysis of change in mean power output from day one to day two showed that, for
women, mean knee extension power output was reduced one day after eccentric exercise in
the PL trial but not in the BCAA trial. (P = 0.02, ES = 0.67). For men, treatment did not
affect change in mean knee extension power output from day one to day two.
Mean knee flexion power
Men had greater mean knee flexion power output than women (P < 0.001, partial η2 =
0.319, Figure 2). The main effect for time was significant (P < 0.001 partial η2 = 0.255);
mean flexion power output was greater on day one than day two and three (P < 0.05). The
main effect for treatment was not significant. The gender × time interaction was significant
(P < 0.001, partial η2 = 0.172); in men, mean knee flexion power output was greater on day
one than day three (P < 0.05). Because a borderline quadratic effect was observed in mean
knee flexion power output for the interaction of gender and time, (Quadratic P = 0.16),
change scores were analyzed separately for men and women. Sub-group analysis of change
in mean power output from day one to day two showed that, for women, mean knee flexion
power output was reduced one day after eccentric exercise in the PL trial but not in the
BCAA trial. (P = 0.03, ES = 0.65). For men, treatment did not affect change in mean knee
flexion power output from day one to day two.
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Creatine phosphokinase activity
Data screening found that creatine phosphokinase enzyme activity was not normally
distributed (Kolmogorov-Smirnov, P < 0.01). Because assumptions of sphericity were not
met (Huynh-Feldt = 0.521), multivariate tests for interactions were used. The main effect for
gender in regard to creatine phosphokinase activity was significant (P < 0.001, partial η2 =
0.372, Figure 3). Men had higher creatine phosphokinase levels than women. The main effect
for time was significant (P < 0.001, partial η2 = 0.612); CPK levels were higher day two and
day three than before or after eccentric exercise on day one. The main effect for treatment
was not significant.
Lactate dehydrogenase activity
Data screening found that lactate dehydrogenase enzyme activity was not normally
distributed (Kolmogorov-Smirnov, P < 0.01). Because assumptions of sphericity were met
(Huynh-Feldt = 0.993), univariate within-subjects tests were used. The main effect for gender
in regard to lactate dehydrogenase activity was significant (P < 0.001, partial η2 = 0.164,
Figure 4); men had higher levels of lactate dehydrogenase than women. The main effects for
treatment and time were not significant.
Leg muscle soreness
Data screening found that leg soreness was not normally distributed (KolmogorovSmirnov, P < 0.01). Because assumptions of sphericity were not met (Huynh-Feldt = 0.644),
multivariate tests for interactions were used. The main effect for time in regard to soreness
was significant (P < 0.001, partial η2 = 0.793, Figure 5). Leg muscle soreness was reported to
be greater on day one after eccentric exercise and day two before and after isokinetic testing
than before eccentric exercise on day one (P < 0.05). Leg muscle soreness was reported to be
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greater on day three both before and after exercise than day two and day one (P < 0.05). The
main effects for gender and treatment were not significant.
Ratings of perceived exertion
Data screening found that RPE were not normally distributed (Kolmogorov-Smirnov,
P < 0.01). Therefore, data were transformed into ranked cases from smallest to highest for all
trials combined. Ranks were then separated by trial number for analysis. Because
assumptions of sphericity were not met (Huynh-Feldt = 0.739), multivariate tests for
interactions were used. The main effect for time in regard to ratings of perceived exertion
was significant (P < 0.001, partial η2 = 0.649). Ratings of perceived exertion were lower after
the first set of eccentric exercise than after the third set, which in turn were lower than the
RPE after the sixth set. The main effects for gender and treatment were not significant.
Side effects
Anecdotally, none of the participants reported any negative side effects from BCAA
supplementation.
DISCUSSION
The purpose of this study was to determine the effects of acute ingestion of BCAA on
muscle function, plasma markers of muscle damage, and subjective evaluation of muscle
soreness after eccentric knee extensions and flexions. To our knowledge, this is the first
study to examine the effects of acute BCAA ingestion on these measures.
The most important finding of the current study was that ingestion of 8 g BCAA
helped maintain mean power output 24 hours after eccentric exercise in middle-aged women.
Mean power output did not decrease from day one to day two for either knee extensions or
flexions in the BCAA trial, while mean knee extension and flexion power output decreased
117
from day one to day two in the PL trial. The calculated effect sizes were of moderate
magnitude. Sugita et al. (33), examined the effects of taking an amino acid mixture (5.6 g,
30% BCAA by weight) b.i.d on muscle performance after eccentric exercise. Peak isometric
muscle torque was greater after intake of amino acids two, three, and six days after eccentric
exercise. These findings suggest that BCAA supplementation may attenuate losses of muscle
performance after eccentric exercise.
Interestingly, acute ingestion of 8 g BCAA did not maintain mean power output in
men. Our results corroborate the findings of others that have shown men and women may
respond differently to eccentric exercise. Sewright et al. (28), reported that women
experienced greater immediate relative strength loss than men after 50 maximal eccentric
contractions of the elbow flexors of the non-dominant arm. Furthermore, a disproportionately
greater number of females than males demonstrated force reductions of > 70% maximal
voluntary contraction (MVC) after eccentric contractions. Men, however, had greater CPK
responses four days after eccentric exercise than women. In our study, men had higher CPK
and LDH levels than women. In contrast to Sewright and colleagues, we observed loss of
muscle torque, power, and total work performed in the knee flexors of men, not women. The
reason for the gender differences in response to eccentric exercise is unknown. Hormonal
differences between men and women, particularly estradiol, may influence the response to
eccentric exercise. Amelink and Bär (1) suggested that estrogen may serve to protect the
female muscle membranes in rats. Another proposed explanation of gender differences in
force or power generation after eccentric exercise is possible differences between men and
women in habitual muscle use. Untrained muscles are more prone to damage than trained
muscles (7). The lower level of physical activity in females (35) may explain their greater
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levels of muscle damage or loss of muscle function after eccentric or novel exercise.
However, in our study, men had less mean power, torque, and total work performed for knee
flexions 48 hours after eccentric exercise than before eccentric exercise. This trend was not
observed in women. While gender differences in response to eccentric exercise may exist, to
our knowledge, the mechanisms have yet to be elucidated. Furthermore, contradicting
findings exist regarding such gender differences.
It is plausible that men and women respond differently to BCAA intake. Men oxidize
leucine at a higher rate than women and also have lower nonoxidative leucine disposal than
women during exercise, suggesting that women have higher rates of protein synthesis than
men during exercise and may therefore experience greater benefit from BCAA
supplementation (19). We observed that mean flexion and extension power output were
maintained when women consumed BCAA. Others studies examining the effects of BCAA
on strength and performance in males found that BCAA may not improve performance.
Leucine supplementation for 19 days did not affect knee extensor strength in elite wrestlers
(23). Leucine supplementation (100 mg/kg bodyweight) before and during a strength training
session did not affect counter movement jump performance in competitive male power
athletes (26). In contrast, leucine supplementation (45 mg/kg body weight) for six weeks
increased relative peak power during a ten-second all-out arm crank test in competitive
outrigger canoeists (9). Interpreting our results in light of these studies previous is difficult,
since our participants were not highly trained athletes.
Acute BCAA supplementation did not affect enzymatic markers of muscle membrane
damage (CPK and LDH) in the current study. Others report that BCAA supplementation may
attenuate exercise-induced muscle damage or muscle proteolysis (8, 20, 25, 34). BCAA (77
119
mg/kg) administered before 60 minutes of knee extensor exercise suppressed endogenous
protein breakdown in men (20). In our study, endogenous protein degradation was not
measured. Longer-term BCAA supplementation (12 g/d, 14 d) reduced CPK and LDH
increases in blood for up to five days after 120 minutes of cycle ergometer exercise at 70%
VO2 max in males (8). An amino acid (6.6 g/d, 30% BCAA, by weight) mixture administered
for one month to middle- and long-distance collegiate track runners significantly reduced
serum CPK levels pre-test to post-test during training (26). BCAA supplementation (12 g/d,
15 d) prevented an increase in urinary urea nitrogen, hydroxyproline, and 3-methylhistidine
20 hours after 600 m crawl stroke swimming competition (34). Furthermore, three weeks of
BCAA supplementation before one week of high-intensity resistance training resulted in
lower serum CPK and cortisol levels and higher serum total testosterone levels during and
after training (29) Conversely, consumption of a carbohydrate/protein drink (23 g whey
protein, 75 g carbohydrate, which contained 5.4 g BCAA) either before or after eccentric
exercise did not affect muscle soreness, loss of muscle strength, or blood CPK levels up to 96
hours after eccentric exercise (37). Jackman et al. (16) found that 7.3 g BCAA
supplementation before and three times after eccentric exercise on the day of exercise and
four times per day for two days after exercise did not affect plasma CPK or serum
interleukin-6, an indirect marker of inflammation. Taken together, the results on the effects
of BCAA supplementation on markers of muscle damage or membrane disruption are
equivocal and may be influenced by length of supplementation period.
Acute BCAA supplementation did not affect subjective evaluation of muscle soreness
in the current study. This finding contradicts others (16, 31). Jackman et al. (16) observed
that males receiving BCAA reported lower knee flexed soreness of the quadriceps 48 and 72
120
hours after eccentric exercise than those receiving placebo. However, BCAA
supplementation did not influence overall soreness or knee extended soreness ratings. Acute
BCAA supplementation (5 g) ingested 15 minutes before seven sets of ten repetitions of
squat exercise decreased muscle soreness in women for four days after exercise and tended to
be lower in males, compared to placebo (31). Shimomura et al. (31) suggested the gender
difference in DOMS may be related to the smaller relative dose of BCAA consumed by
males than females. Males ingested 77 mg/kg bodyweight BCAA, on average, whereas
females ingested 92 mg/kg bodyweight BCAA. In our study, male participants consumed
approximately 90.5 mg/kg bodyweight BCAA, whereas female participants consumed
approximately 113.6 ml/kg bodyweight. Additional examination of the effects of relative
BCAA dose on subjective appraisal of muscle soreness is warranted. Delayed-onset muscle
soreness may theoretically influence exercise program adherence. According to the Hedonic
Theory of Motivation and Affective Responses to Exercise, persons seek things that
maximize pleasure and avoid those that cause displeasure. Furthermore, affective responses
to a behavior may predict future behaviors (17). If an untrained person begins a new exercise
regimen and experiences intense muscle soreness, it is possible that he or she ceases program
adherence because of the displeasure experienced from the associated muscle soreness.
Supplements possessing anti-soreness properties may therefore become central in the effort
to promote health by increasing physical activity program adherence.
The major limitation of the current study is that participants’ diets were not recorded
and replicated. It is possible that potential prophylactic effects of acute BCAA
supplementation on muscle soreness, muscle function, or membrane disruption were
121
prevented from dietary intake differences between trials. A follow-up study in which diet is
recorded and replicated is highly warranted.
PRACTICAL APPLICATIONS
Significant loss of muscle strength and power may occur during periods of muscle
soreness. We observed that acute BCAA supplementation may attenuate loss of muscle
function and may maintain muscle performance after eccentric exercise in middle-aged
women. This finding may be important for strength and conditioning professionals working
with middle-aged women involved in sports. In men, mean power, peak torque, and total
work performed during knee flexions were lower 48 hours after eccentric exercise, regardless
of treatment. We did not observe these losses of muscle function in women.
Acknowledgments
We would like to thank all participants who volunteered for this study. We would
also like to thank the reviewers for their time and consideration involved in the review of this
manuscript. This study was funded by a grant from Otsuka Pharmaceutical Co., Ltd., Japan.
The authors declare that they have no conflicts of interest. The results of the present study do
not constitute endorsement of the product by the authors or the National Strength and
Conditioning Association.
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Figure Legends
Table 1. Participant characteristics. All values are means ± SE.
Figure 1. Mean (± SE) extension power. BCAA indicates branched-chain amino acids; PL,
placebo. Men significantly different than women (main effect; P < 0.001).
Figure 2. Mean (± SE) flexion power. BCAA indicates branched-chain amino acids; PL,
placebo. * Men significantly different than women (P < 0.001). † Significantly different than
day 1 (P < 0.05).
Figure 3. Mean (± SE) plasma creatine phosphokinase levels. BCAA indicates branchedchain amino acids; PL, placebo. * Men significantly different than women (main effect; P <
0.001). † Significantly different than day 1 pre and post (P < 0.05).
Figure 4. Mean (± SE) plasma lactate dehydrogenase levels. BCAA indicates branched-chain
amino acids; PL, placebo. * Men significantly different than women (main effect; P < 0.001).
Figure 5. Mean (± SE) leg soreness score. BCAA indicates branched-chain amino acids; PL,
placebo. a, b, c Time points with different superscripts are different (P < 0.05).
Table 1. Participant characteristics. All values are means ± SE.
Men (n = 20)
Women (n = 25)
Age (yr)
47.1 ± 1.4
48.6 ± 1.2
Height (cm)
177.8 ± 1.7*
162.1 ± 1.2
Mass (kg)
88.4 ± 2.9*
70.4 ± 2.8
BMI (kg/m2) 27.9 ± 0.9
26.8 ± 1.0
FFM (kg)
46.7 ± 1.4
68.3 ± 1.9*
Age, height, mass, body mass index (BMI), and fat-free mass (FFM). *Significantly different
from women (P < 0.001).
ExtensionPower (watts)
ExtensionPower (watts)
126
110
108
106
104
102
100
98
96
94
92
90
180
178
176
174
172
170
168
166
164
162
160
158
156
154
152
150
Women
Placebo
BCAA
Men*
Placebo
BCAA
Day 1
Day 2
Day 3
Figure 1. Mean (± SE) mean extension power. * Men significantly different than women
(main effect; P < 0.001).
127
FlexionPower (watts)
54
Women
Placebo
BCAA
52
50
48
46
44
42
FlexionPower (watts)
40
90
86
82
78
74
70
66
62
58
54
50
Men*
Placebo
BCAA
Day 1
Day 2
Day 3
Figure 2. Mean (± SE) mean flexion power. * Men significantly different than women (P <
0.001). † Significantly different than day 1 (P < 0.05)
Creatinephosphokinase(U/L)
1000
900
800
700
600
500
400
300
200
100
0
Creatinephosphokinase(U/L)
128
1000
900
800
700
600
500
400
300
200
100
0
Women
Placebo
BCAA
Men*
Placebo
BCAA
Pre
Day 1
Post
Day 2
Day 3
Figure 3. Mean (± SE) plasma creatine phosphokinase levels. * Men significantly different
than women (main effect; P < 0.001). † Significantly different than day 1 pre and post (P <
0.05).
Lactatedehydrogenase(U/L)
110
100
90
80
70
60
50
40
30
20
10
0
Lactatedehydrogenase(U/L)
129
110
100
90
80
70
60
50
40
30
20
10
0
Women
Placebo
BCAA
Men*
Placebo
BCAA
Pre
Day 1
Post
Day 2
Day 3
Figure 4. Mean (± SE) plasma lactate dehydrogenase levels. * Men significantly different
than women (main effect; P < 0.001).
130
SubjectiveLegSoreness
5
Women
4
Placebo
BCAA
3
2
1
0
SubjectiveLegSoreness
5
Men
4
Placebo
BCAA
3
2
1
0
a
b
b
b
c
c
Pre
Post
Pre
Post
Pre
Post
Day 1
Figure 5. Mean (± SE) leg soreness score.
(P < 0.05).
Day 2
a, b, c
Day 3
Time points with different letters are different
131
ACKNOWLEDGEMENTS
I would like to thank Dr. Doug King for serving as my major professor. His guidance,
patience, mentorship, and friendship have been greatly valued during my Ph.D.
matriculation. Thank you for this opportunity, Boss!
I am also thankful for the assistance, guidance, and friendships I have received from
my other POS committee members – Dr. Ruth Litchfield, Dr. Amy Welch, Dr. Warren
Franke, and Dr. Rick Sharp. I have learned a great deal about teaching and research from
them, for which I am extremely grateful.
A special thanks to Dr. Walter Hsu and his graduate students Ahmed Soliman and
Dai Tan Vo. Your assistance with the hormone assays is sincerely appreciated.
I thank all those who helped with collection of data – Dr. David Senchina, Hector
Angus, Rachel Bell, Bret Fox, Mark Christiani, Erin Taylor, Erin Josephson, Jenn Pieper,
Sade Grant, Allie Ladd, Collin Kilburg, Adam Becker, Haley Risdal, and Hannah Singletary.
I would also like to thank Lesley Hawkins, Jody Burdick, Pat Mize, and Ron Leibold
for all their help over the past several years.
I sincerely thank Mrs. Pease for financially supporting this dissertation research
through the Pease Family Doctoral Research Award.
I would also like to thank my family – Mom (Mary Ann), Dad (Tom), and younger
brothers Zach, Issiah and Elijah. Their unending support and encouragement to pursue my
goals and dreams is thanked, though words will not justify how grateful I am to them.
Finally, my best friend and wife, Christie– thank you dearly for your support,
friendship, companionship, love and understanding during my graduate studies. Also thank
you for being such a wonderful mother to my beautiful daughter Zoe.