J Appl Physiol 115: 785–793, 2013.
First published June 20, 2013; doi:10.1152/japplphysiol.00445.2013.
Improvements in exercise performance with high-intensity interval training
coincide with an increase in skeletal muscle mitochondrial content
and function
Robert Acton Jacobs,1,2,3 Daniela Flück,1,2 Thomas Christian Bonne,4 Simon Bürgi,2
Peter Møller Christensen,4 Marco Toigo,1,2,5 and Carsten Lundby1,2
1
Zurich Center for Integrative Human Physiology (ZIHP), Zurich, Switzerland; 2Institute of Physiology, University of Zurich,
Zurich, Switzerland; 3Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland: 4Department of Exercise
and Sport Sciences, University of Copenhagen, Copenhagen, Denmark; and 5Exercise Physiology, Institute of Human
Movement Sciences, ETH Zurich, Zurich, Switzerland
Submitted 11 April 2013; accepted in final form 18 June 2013
interval training; sprint training; HIT; oxygen extraction; mitochondria
HIGH-INTENSITY INTERVAL TRAINING
(HIT) has repeatedly demonstrated its proficiency as a resourceful training modality to
improve exercise performance. Over the course of six HIT
training sessions, typically over 2 wk time, marked improvements in exercise capacity have been observed in normal
healthy individuals (3, 9, 10, 41, 63), diseased populations (39,
69), and even trained athletes (11, 24, 37, 38, 62). A compre-
Address for reprint requests and other correspondence: C. Lundby, Institute
of Physiology, ZIHP, Univ. of Zürich, Office 23 H 6, Winterthurerstrasse 190,
8057 Zürich, Switzerland (e-mail: [email protected]).
http://www.jappl.org
hensive mechanistic explanation for these rapid improvements
in exercise performance is however lacking.
One single session of HIT sufficiently stimulates transcriptional activation of mitochondrial biogenesis (4, 20, 40, 49),
even apparent in highly trained athletes (53), and remains
evident following the 2 wk of HIT (25, 41). This transcriptional
stimulus translates into phenotypic changes in mitochondrial
protein expression and activity, including proteins involved in
fat oxidation, tricarboxylic acid (TCA) cycle and/or the electron transport chain (9, 10, 25, 39 – 41, 49, 63). These static
measures of biochemical expression within the muscle indirectly suggest enhanced oxidative potential, however the direct
assessment of respiratory capacity, substrate control, and fatigue resistance in the skeletal muscle is lacking. Measurable
alterations in mitochondrial protein expression (64) do not
necessarily translate into functional changes (26), and alternatively functional alterations in mitochondria can occur independent from a measured change in protein expression (32).
Thus interpreting observable changes in isolated protein expression as an adaptation of complex and integrative subcellular systems may be inaccurate. Regardless, these adaptations
in the skeletal muscle are primarily used to explain the improvements in exercise performance (18).
Mass-specific respiratory capacity of the skeletal muscle is
the single most predictive physiological variable of endurance
performance (31). Therefore, a mitochondria-centric mechanistic explanation for the improvements in endurance performance following six sessions of HIT (10, 38, 39, 41, 62, 63) is
most likely appropriate. However, six sessions of HIT have
also repeatedly demonstrated a proficiency for improving measures of maximal exercise performance such as maximal oxygen uptake (V̇O2peak) and peak power output via graded exercise tests (3, 38, 39, 63, 69), although a measurable improvement does not always occur (10). Peak power output obtained
during whole body exercise is largely controlled by a limitation
in oxygen delivery to the locomotor muscles (2) and accordingly is best predicted by the capacity for oxygen transport as
well as the ability for the working muscle to extract oxygen
(31, 47, 55). Enhanced oxidative capacity of the skeletal
muscle alone would not be expected to explain these improvements in measures of maximal exercise performance as the
respiratory capacity of the skeletal muscle exceeds maximal
oxygen delivery to the working muscles (5). Rather these
improvements in exercise would most likely be attributed to an
8750-7587/13 Copyright © 2013 the American Physiological Society
785
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Jacobs RA, Flück D, Bonne TC, Bürgi S, Christensen PM, Toigo M,
Lundby C. Improvements in exercise performance with high-intensity interval training coincide with an increase in skeletal muscle mitochondrial
content and function. J Appl Physiol 115: 785–793, 2013. First published
June 20, 2013; doi:10.1152/japplphysiol.00445.2013.—Six sessions of
high-intensity interval training (HIT) are sufficient to improve exercise capacity. The mechanisms explaining such improvements are
unclear. Accordingly, the aim of this study was to perform a comprehensive evaluation of physiologically relevant adaptations occurring
after six sessions of HIT to determine the mechanisms explaining
improvements in exercise performance. Sixteen untrained (43 ⫾ 6
ml·kg⫺1·min⫺1) subjects completed six sessions of repeated (8 –12)
60 s intervals of high-intensity cycling (100% peak power output
elicited during incremental maximal exercise test) intermixed with 75
s of recovery cycling at a low intensity (30 W) over a 2-wk period.
Potential training-induced alterations in skeletal muscle respiratory
capacity, mitochondrial content, skeletal muscle oxygenation, cardiac
capacity, blood volumes, and peripheral fatigue resistance were all
assessed prior to and again following training. Maximal measures of
oxygen uptake (V̇O2peak; ⬃8%; P ⫽ 0.026) and cycling time to
complete a set amount of work (⬃5%; P ⫽ 0.008) improved. Skeletal
muscle respiratory capacities increased, most likely as a result of an
expansion of skeletal muscle mitochondria (⬃20%, P ⫽ 0.026), as
assessed by cytochrome c oxidase activity. Skeletal muscle deoxygenation also increased while maximal cardiac output, total hemoglobin, plasma volume, total blood volume, and relative measures of
peripheral fatigue resistance were all unaltered with training. These
results suggest that increases in mitochondrial content following six
HIT sessions may facilitate improvements in respiratory capacity and
oxygen extraction, and ultimately are responsible for the improvements in maximal whole body exercise capacity and endurance
performance in previously untrained individuals.
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Mechanisms Explaining Exercise Improvements with HIT
increase in oxygen delivery to and/or greater oxygen extraction
in the lower limbs (12, 57, 59, 60).
Accordingly, the aim of the present study was to perform a
comprehensive evaluation of the physiological adaptations,
ranging from cardiovascular to skeletal muscle properties,
following six sessions of HIT in untrained young adults and
determine the mechanisms explaining rapid improvements in
exercise performance. We hypothesize that improvements in
respiratory capacity of the skeletal muscle will parallel any
marked increases of endurance capacity (31), while a greater
maximal cardiac output and/or improved oxygen extraction of
the locomotor muscles will correspond to improvements in
V̇O2peak and peak power output (12, 57, 60).
MATERIALS AND METHODS
Table 1. Subject characteristics
Weight, kg
Total body fat, %
V̇O2peak, l/min
Hbmass, g
[Hb], g/dl
Hct, %
RCV, ml
PV, ml
TBV, ml
Pre-HIT
Post-HIT
P Value
77.1 ⫾ 2.1
20.5 ⫾ 1.3
3.3 ⫾ 0.2
854 ⫾ 30
15.1 ⫾ 0.2
43.4 ⫾ 0.4
2,500 ⫾ 97
3,286 ⫾ 141
5,786 ⫾ 233
76.9 ⫾ 2.2
20.0 ⫾ 1.4
3.6 ⫾ 0.1
850 ⫾ 29
14.9 ⫾ 0.1
43.3 ⫾ 0.4
2,481 ⫾ 96
3,284 ⫾ 108
5,765 ⫾ 199
0.468
0.048
0.031
0.796
0.432
0.652
0.714
0.983
0.865
Measurements are presented as means ⫾ SD and represent baseline values
and those determined following 6 high-intensity interval training (HIT) sessions, lasting a total of 2 wk. V̇O2peak, maximal oxygen uptake; Hbmass, total
hemoglobin mass; [Hb], hemoglobin concentration; Hct, hematocrit; RCV, red
blood cell volume; PV, plasma volume; TBV, total blood volume.
Jacobs RA et al.
Mean V̇O2peak was determined as the highest value averaged over 30
s for each subject. The subjects then were allowed a 1-h rest before
beginning a time trial. Following the rest each subject performed a
time trial in which they completed a set workload as quickly as
possible. The criteria of the time trial was completing a set absolute
workload that was relative to each individual subject’s maximal
exercise performance at baseline, determined as 25% more work than
was completed during the baseline incremental cycling test. This
workload determination was selected for the time trial exercise tests
because it 1) controlled for differences in baseline aerobic capacity
across subjects, and 2) normalized the time of completion of all
baseline tests at ⬃20 min (1,306 ⫾ 210 s). This allowed for a more
standardized test of endurance across a semiheterogeneous group of
subjects. All time trials were performed on an electronically braked
ergometer to become familiar with these performance tests. Each
subject could increase or decrease resistance manually and/or via their
cadence as desired to complete the predetermined workload. Subjects
were instructed to complete the tests as quickly as possible and were
provided no temporal or physiological feedback. The only feedback
provided was their real-time workload, cadence, and remaining workload until completion. Exercise duration and power output were
recorded upon completion of each test.
Training. The specific HIT protocol used in the present study was
derived from previous work (41). Briefly, following baseline testing
all subjects began the training protocol, which consisted of six
sessions over 2 wk (training every 2–3 days). Each training session
consisted of repeated 60 s efforts of high-intensity cycling at a
workload that corresponded to the peak power achieved during the
incremental cycling test (249 ⫾ 52 W). These high-intensity intervals
were intermixed with 75 s of cycling at a low intensity (30 W) for
recovery. Subjects completed 8 high-intensity intervals during the first
two training sessions, 10 intervals during the third and fourth sessions,
and 12 intervals on the final two sessions. A 3-min warm-up at 30 W
was performed each day prior to training. The time commitment
during each training session ranged from 21 min for the first two
sessions, 25 min for the 3rd and 4th sessions, and 30 min for the final
two sessions. The total time spent training over the 2-wk span,
including warm-up and recovery, was under 3 h. All subjects completed all training sessions.
Skeletal muscle sampling. Skeletal muscle biopsies were obtained
under standardized conditions from the vastus lateralis muscle at
baseline and again after the 2-wk, six-session regimen of HIT.
Samples were collected under local anesthesia (1% lidocaine) of the
skin and superficial muscle fascia, using the Bergström technique with
a needle modified for suction. The biopsy was immediately dissected
free of fat and connective tissue and divided into sections for measurements of mitochondrial respiration. All biopsies were taken ⬃48 h
following the last bout of exercise.
Skeletal muscle preparation. The biopsy was sectioned into parts to
measure mitochondrial respiration as previously described (26 –28,
30, 32, 50). Respiration measurements were performed in mitochondrial respiration medium 06 (MiR06; MiR05 ⫹ catalase 280 IU/ml).
Measurements of O2 consumption were performed at 37°C using the
high-resolution Oxygraph-2k (Oroboros, Innsbruck, Austria). Standardized instrumental and chemical calibrations were performed as
described previously (32). Oxygen flux was resolved by software
allowing nonlinear changes in the negative time derivative of the
oxygen concentration signal (DatLab, Oroboros, Innsbruck, Austria).
All respirometric analyses were done in duplicate and were carried out
in a hyperoxygenated environment to prevent any potential oxygen
diffusion limitation with oxygen concentration within the chamber
ranging between 250 and 420 nmol/ml.
Respiratory titration protocol. The titration protocol was specific to
the examination of individual aspects of respiratory control through a
sequence of coupling and substrate states induced via separate titrations. This titration protocol was modified from previous protocols
where they are described in detail (29, 32). All titrations were added
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Ethical approval. These experimental protocols involving human
subjects were approved by the Ethical committee for the ETH Zürich
(EK 2011-N-24), in accordance with the declaration of Helsinki. Prior
to the start of the experiments, informed oral and written consents
were obtained from all participants.
Subjects. Sixteen untrained (V̇O2peak 43 ⫾ 6 ml·kg⫺1·min⫺1) adult
(aged 27 ⫾ 3 yr, height 181 ⫾ 6 cm) male subjects participated in this
study (data presented as means ⫾ SD). No subject regularly engaged
in either 1) a structured exercise training program, or 2) high-intensity
exercise. Aerobic activity prior to commencement of the study was
limited to less than 3 h of light aerobic activity per week. All subjects
were medication free throughout the duration of the study. All
subjects were scanned for body composition in a dual-energy X-ray
absorptiometer (Lunar iDXA, GE Healthcare, Madison, WI) prior to
exercise training and again following completion of training (Table 1)
with time of the day (morning) when scanned standardized for all
subjects.
Experimental design. The experimental design consisted of familiarization period, baseline testing, a 2-wk exercise training intervention, and posttraining measurements.
Familiarization, baseline, and post-training exercise testing. Subjects initially performed an incremental cycling test to volitional
fatigue on an electronically braked cycle ergometer (Monark E839,
Varberg, Sweden) to determine V̇O2peak and peak power output using
an online gas collection system (Innocor, Innovision, Odense, Denmark) where O2 and CO2 concentration in the expired gas was
continuously measured and monitored as breath-by-breath values. The
gas analyzers and the flowmeter of the applied spirometer were
calibrated prior to each test. Subjects began exercise at three consecutive 5-min workloads, increasing by 50-W increments, beginning
with 50 W and finishing with 150 W. From that point the workload
was increased by 30 W every 90 s thereafter until volitional fatigue.
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Mechanisms Explaining Exercise Improvements with HIT
Jacobs RA et al.
787
pulmonary uptake of a blood-soluble gas is proportional to pulmonary
blood flow (34). For each measurement subjects rebreathed a test gas
mixture consisting of 5% blood soluble N2O, 1% insoluble sulfur
hexafluoride (SF6), and 94% O2. This specific combination of gasses
were mixed together along with ambient air into a rebreathing bag
prior to each measurement. The Innocor takes into account the tidal
volume and V̇O2 during exercise along with the ratio of the test gas to
ambient air, and bag volume to allow unrestricted ventilation during
the rebreathing maneuver. For measurements the subjects were
switched from breathing room air to breathing from the closed circuit,
rebreathing the test gas mixture while photo-acoustic gas analyzers
continuously quantified the gas concentrations in the rebreathing
circuit. Pulmonary N2O uptake was determined by the decrease in
N2O concentration over three consecutive expirations when complete
mixture is obtained between residual pulmonary air and the gas in the
rebreathing bag as indicated by the insoluble gas SF6. Prior to each
exercise test, including those during the familiarization period, the
subjects were instructed on how to perform the rebreathing and then
conducted two to three maneuvers to practice.
Blood characteristics. Total hemoglobin mass (Hbmass) was measured as previously described (31), using a modified version of a
carbon monoxide (CO) rebreathing technique. Each subject would
come to the lab and first rest for 20 min in a semirecumbent position.
Then they breathed 100% O2 for 5 min to flush the nitrogen from the
airways. Thereafter, 2 ml of blood was sampled from an antecubital
vein via a 20-G catheter and analyzed immediately in quadruplicate
for 1) percent carboxy-Hb (%HbCO) and Hb concentration ([Hb])
using a hemoximeter (ABL800, Radiometer, Copenhagen, Denmark);
and 2) hematocrit with the micromethod (4 min at 13,500 rpm). The
breathing circuit (previously flushed with O2) was then closed, and a
bolus (1.5 ml/kg) of 99.997% chemically pure CO (CO N47, Air
Liquide, Paris, France) was administered into the now closed rebreathing apparatus. The subject rebreathed this gas mixture for 10
min. At the end of the rebreathing period, an additional 2 ml blood
sample was obtained and analyzed as before. The change in %HbCO
between first and second measurement was used to calculate Hbmass,
taking into account the amount of CO that remained in the rebreathing
circuit at the end of the procedure (2.2%) (8). Total red blood cell
volume (RCV), total blood volume (TBV), and plasma volume were
derived from measures of Hbmass, [Hb], and hematocrit (8). The same
researcher performed all CO rebreathing tests. Baseline and post-HIT
training values reported here correspond to the average of duplicate
measurements conducted on separate days within these testing sessions. The coefficient of variation for Hbmass, assessed from duplicate
measures at baseline and expressed as the percent typical error (i.e.,
SD of difference scores/兹2), was 2.5%.
Peripheral fatigue resistance. The magnitude of peripheral quadriceps fatigue was quantified via supramaximal magnetic stimulation
of the femoral nerve, with the exercise-induced reduction in potentiated quadriceps twitch force (Qtw,pot) assessed prior to exercise and
again 30 s after termination of exercise following both the graded
exercise and time trial tests.
Briefly, subjects sat semirecumbent on a chair so that their knee
joint angle was between 85 and 95 degrees. A Magstim Rapid (220)
stimulator (Magstim, Whitland, UK), with a figure-of-eight coil, was
applied to stimulate the femoral nerve. The evoked quadriceps twitch
force was measured using a strain gauge that was fixed to a rigid pole
of the chair on one end with the other end connected to a noncompliant strap placed just superior to the malleolus of each subject’s
dominant leg. Electrodes were placed in a bipolar configuration over
the center of the vastus lateralis muscle to record magnetically evoked
compound action potentials. Active electrodes were placed over the
motor point of the muscle, quadriceps, and the reference electrode
placed in an electrically neutral site, elbow.
For evaluation of locomotor muscle strength each subject was
asked to perform six 5-s maximal voluntary contractions (MVC).
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in series as presented. Leak respiration in absence of adenylates (LN)
was induced with the addition of malate (2 mM) and octanoyl
carnitine (0.2 mM). Maximal electron flow through electron transferring-flavoprotein (ETF) and fatty acid oxidative capacity (PETF) was
determined following the addition of ADP (5 mM). Submaximal state
3 respiratory capacity specific to CI (PCI) was induced following the
additions of pyruvate (5 mM) and glutamate (10 mM). Maximal state
3 respiration, oxidative phosphorylation capacity (P), was then induced with the addition of succinate (10 mM). As an internal control
for compromised integrity of the mitochondrial preparation, the mitochondrial outer membrane was assessed with the addition of cytochrome c (10 ␮M). There was no evidence of any compromised
mitochondrial membrane integrity across samples measured at baseline with the titration of exogenous cytochrome c (86.5 ⫾ 15.6 to 86.6 ⫾
15.5 pmol O2·min⫺1·mg wet wt⫺1, P ⫽ 0.611) or following 2 wk of
HIT exercise training (99.8 ⫾ 23.1 to 100.3 ⫾ 23.2 pmol
O2·min⫺1·mg wet wt⫺1, P ⫽ 0.259). Oligomycin was added inhibiting ATP synthase to achieve oligomycin-induced leak respiration
(LOMY). Phosphorylative restraint of electron transport was assessed
by uncoupling ATP synthase (complex V) from the electron transport
system with the titration of the proton ionophore, carbonyl cyanide
p-(trifluoromethoxy) phenylhydrazone (FCCP; steps of 0.5 ␮M)
reaching electron transport system (ETS) capacity. Rotenone (0.5
␮M) and antimycin A (2.5 ␮M) were added, in sequence, to terminate
respiration by inhibiting CI and complex III (cytochrome bc1 complex), respectively. With CI inhibited, electron flow specific to CII
(PCII) can be measured. Prior uncoupling with FCCP has no effect on
PCII (32), as individual electron input to CII does not saturate the
Q-cycle. Inhibition of respiration with antimycin A then allows for the
determination and correction of residual oxygen consumption, indicative of non-mitochondrial oxygen consumption in the chamber.
Finally, ascorbate (2 mM) and TMPD (0.5 mM) were simultaneously
titrated into the chambers to assess cytochrome c oxidase (COX),
complex IV, activity. TMPD and ascorbate are redox substrates that
donate electrons directly to COX and activity was measured by
picomoles O2 per minute per mg wet weight. COX activity has been
shown to strongly correlate with mitochondrial volume density and
total cristae area (both measured via transmission electron microscopy) in addition to respiratory capacity (36).
Skeletal muscle oxygenation. Absolute changes in concentrations
(␮mol/l) of deoxygenated hemoglobin (Hb) and myoglobin (Mb)
(⌬HHbMb), in addition to the skeletal muscle oxygenation index
(⌬mStO2), the ratio of oxygenated to total tissue Hb and Mb, was
obtained from the vastus lateralis muscle via spatially resolved nearinfrared spectroscopy (NIRS; NIRO-200NX, Hamamatsu, Japan) with
respect to an initial value arbitrarily set equal to zero. NIRS provides
a noninvasive means to continuously and simultaneously monitor
oxygenation status of the hemoglobin in the microvasculature and
myoglobin in the skeletal muscle reflecting the balance between
oxygen consumption (drive to reduce oxygenation) and delivery
(drive to maintain oxygenation). Measures of ⌬HHbMb (15) or
⌬mStO2 (6) values have been used to serve as a surrogate measure of
oxygen extraction. Tissue oxygenation parameters were obtained
using one light-emitting diode (LED; 735, 810, and 850 nm) and two
recording sensors spaced 0.8 cm apart. The source/detector separation
was 3 cm. All measurements are based on the diffusion approximation
for a highly scattering semi-infinite homogeneous media. The decrease in reflected light is measured as a function of the source
detector separation distance and the effective light attenuation coefficient can be estimated. Assuming wavelength dependence of the
reduced scattering coefficient, the spectral shape of the absorption
coefficient can be calculated and tissue saturation estimated.
Cardiac output. Maximal cardiac output (Qmax) was determined
using the Innocor (Innovision, Odense, Denmark) during the maximal
incremental exercise test. Assessment of Q with the Innocor is based
on rebreathing technique and derives Q from pulmonary uptake of the
inert gas nitrous oxide (N2O). This rebreathing technique assumes that
•
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Mechanisms Explaining Exercise Improvements with HIT
RESULTS
Exercise capacity and body composition. Following six
sessions of HIT over 2 wk absolute and relative measures of
V̇O2peak increased by 7.9% (P ⫽ 0.031) and 8.2% (P ⫽ 0.026),
respectively, while peak power output increased by 7% (P ⫽
0.002, Fig. 1A). Endurance capacity also improved, as the
subjects were able to complete the same absolute workload ⬃1
min faster (4.4% decrease in time, P ⫽ 0.008) with a 5.1%
improvement in average power output (P ⫽ 0.013; Fig. 1B).
Although the absolute mean power output for the time trials
improved, the relative mean power output as a percent of
maximal power output was the same at baseline as it was after
HIT (with mean ⫾ SD of 62.1 ⫾ 8.9 and 60.6 ⫾ 6.7,
respectively, P ⫽ 0.367). Alterations in total body mass were
negligible with training; however, total body fat percentage
decreased (P ⫽ 0.048) with training (Table 1).
Skeletal muscle respiratory capacity. All respiratory states
measured improved in response to the six HIT sessions except
for submaximal state 3 respiration specific to mitochondrial
complex II (PCII; Fig. 2A). Mitochondrial content as assessed
by COX activity (Fig. 2B), an established biomarker of mitochondrial volume density in human skeletal muscle (36), also
increased with training (P ⫽ 0.026). All measured increases in
respiratory capacity were silenced when normalizing massspecific respirometric values to measures of mitochondrial
content (Fig. 2B), suggesting that the improvements in the
oxidative capacity of the skeletal muscle were due to an
expansion of the mitochondrial network as opposed to qualitative alterations in function.
Tissue oxygenation. NIRS assessed parameters of skeletal
muscle oxygenation changed with HIT training. Measures of
⌬mStO2 and ⌬HHbMb decreased (P ⫽ 0.037) and increased
(P ⫽ 0.001), respectively, at peak power output (Table 2).
Average values of ⌬mStO2 obtained during the time trial also
Jacobs RA et al.
Fig. 1. Alterations in exercise capacity with high-intensity interval training
(HIT). A: changes in maximal oxygen uptake (V̇O2peak) and maximal power
output achieved during the incremental exercise tests are presented on the left
and right y-axes, respectfully. B: improvements during the time trial and the
average power output during the time trial are presented on the left and right
y-axes, respectfully. The white- and gray-filled box plots represent baseline
and post-HIT measurements, respectively. Difference from pre- to postvalues
of *P ⱕ 0.05, **P ⱕ 0.01.
decreased (P ⫽ 0.022) while ⌬HHbMb showed a tendency
(P ⫽ 0.062) to increase. These alterations in skeletal muscle
oxygenation observed during each matched relative intensity
(100% and 60%, respectively) are not surprising as peak power
output and average power output produced during the time trial
both increased with training (Fig. 1). Alterations in skeletal
muscle oxygenation at the same absolute intensities, however,
were also observed to change as ⌬mStO2 at 150, 180, 210, and
240 W diminished (Fig. 3A) and ⌬HHbMb increased at 100,
150, 180, 210, 240, and 270 W (Fig. 3B).
Cardiovascular parameters. Maximal cardiac output failed
to improve after two wk of HIT (⌬ 0.001%, P ⫽ 0.965; Fig. 4).
Similarly, Hbmass, [Hb], Hct, RCV, PV, and TBV were all
unaltered after six HIT sessions (Table 1).
Peripheral fatigue resistance. The change in Qtw,pot and
MVC prior to and following exercise, regardless of exercise
test, was no different when comparing baseline measures to
those obtained following HIT (Fig. 5, A and B).
DISCUSSION
In the present study we performed an evaluation of physiological adaptation to six sessions of HIT in untrained young
adults in attempt to determine the mechanisms explaining
improvements in exercise performance with this abbreviated
training modality. Improvements in V̇O2peak, peak power output, and time trial performance were observed over the 2-wk
span (Fig. 1). These improvements occurred concurrently with
an increase in skeletal muscle respiratory capacity (Fig. 2) and
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Following the 3rd, 4th, and 5th MVC the femoral nerve was stimulated 3 s into the contraction to determine the superimposed twitch
(TWS). Also, 3 s after each MVC, three stimulations were made, each
separated by 3 s, to determine the twitch force (Qtw,pot). The TWS
force was related to the Qtw,pot recorded 3 s after the MVC and termed
percent voluntary muscle activation: %VA ⫽ [1 ⫺ (TWS/Qtw,pot)] ⫻
100 (45).
Following both the maximal incremental exercise test and time
trial, subjects were promptly assisted back into the chair and repositioned as they were previously. The procedure described above was
repeated 30 s after the cessation of exercise. This protocol was used
to assess central (neuromuscular) vs. peripheral (skeletal muscle)
fatigue following each respective exercise test. To determine whether
nerve stimulation was supramaximal, a MVC was followed by three
single twitches at 90, 95 and 100% of maximal stimulator power
output in a nonfatigued and fatigued status.
Data analysis. For all statistical evaluations, a P value of ⬍0.05
was considered significant. Two different statistical models were used
for analysis (SPSS Statistics 17.0, SPSS, Chicago, IL). Paired samples
t-tests were used to test the null hypothesis stating no difference
between all baseline measures and those obtained following 2 wk of
HIT exercise training. The sequential respiratory capacities of P
before and following the titration of exogenous cytochrome c to test
mitochondrial membrane integrity were analyzed by a one-way
ANOVA on repeated measurements. Mass-specific respiration was
normalized to COX activity [(respiratory state/COX) ⫻ 100] and
mitochondrial-specific respiratory capacities are presented as a percent of COX.
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Mechanisms Explaining Exercise Improvements with HIT
•
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Jacobs RA et al.
Table 2. Skeletal muscle oxygenation during exercise
Pre MAX
Post MAX
Pre TTavg
Post TTavg
63.4 ⫾ 6.7 56.4 ⫾ 9.3*
66.0 ⫾ 6.8 59.6 ⫾ 12.2*
⌬mStO2, %
⌬HHbMb, ␮mol/l 15.5 ⫾ 19.3 36.5 ⫾ 27.0*** 33.2 ⫾ 16.3 60.6 ⫾ 39.7†
Values are presented as absolute changes, means ⫾ SE, from an initial value
obtained prior to each respective exercise test that was arbitrarily set equal to
zero. Near-infrared spectroscopy derived measures of skeletal muscle oxygenation (⌬mStO2) and deoxygenated hemoglobin and myoglobin (⌬HHbMb) at
peak power output achieved during an incremental exercise test (MAX) and
averaged throughout a set-workload time trial (TT) prior to (Pre) and following
(Post) 6 sessions of high-intensity training (HIT). Difference between corresponding pre and post HIT measurements: *P ⱕ 0.05, ***P ⱕ 0.001. †A trend
toward significance of P ⫽ 0.062.
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Fig. 2. Changes in mitochondrial content and function. Mass-specific respirometric measurements (A), cytochrome c oxidase (COX) activity (B), and
mitochondrial-specific respirometric values (C). LN, respiration in absence of
adenylates; PETF, capacity for fatty acid oxidation; PCI, submaximal state 3
respiration through complex I; P, maximal state 3 respiration - oxidative
phosphorylation capacity; LOMY, oligomycin-induced leak respiration; ETS,
electron transport system capacity; PCII, submaximal state 3 respiration
through complex II. The white and grey filled bars represent baseline and
post-HIT measurements, respectively. Values are presented as means ⫾ SE.
Difference between corresponding pre-and post-HIT measurements: *P ⱕ
0.05, **P ⱕ 0.01.
greater leg deoxygenation (Fig. 3). Moreover, the improvements in exercise performance occurred independent from any
alterations in maximal cardiac capacity (Fig. 4) or blood
characteristics, specifically oxygen-carrying capacity and total
blood volume (Table 1). Together these data empirically corroborate previous findings and postulates stating that improvements in exercise performance after six sessions of HIT over
the span of 2 wk are primarily attributed to enhanced oxidative
potential in the skeletal muscle.
Discernible improvements in exercise capacity have been
observed after only six HIT training sessions in nonhealthy
populations (39, 69), normal healthy individuals (3, 9, 10, 41,
63), and even trained athletes (11, 24, 37, 38, 62). We also
observed marked improvements in V̇O2peak, peak power output,
and time trial completion (Fig. 1).
Here the results indicate that a greater skeletal muscle
oxidative capacity contributes to the improvements in endurance performance observed (Fig. 1B). Our measured improvements in endurance capacity are in line with previous reports of
improvement (10, 38, 39, 41, 62, 63). Congruent with this
premise is the fact that mass-specific respiratory capacity is the
strongest determining factor of endurance performance in
highly trained athletes (31) as well as in race horses (65). It is,
however, not clear exactly how a greater oxidative capacity in
human skeletal muscle may improve V̇O2peak and peak power
output as was observed in this (Fig. 1A) as well as previous
studies (3, 38, 39, 63, 69).
Measures of maximal whole body exercise capacity are
largely controlled by a limitation in oxygen delivery to the
locomotor muscles (2) with respiratory capacity of the skeletal
muscle able to outstrip maximal oxygen delivery to the working muscles (5). Traditional endurance training improves maximal incremental exercise capacity in untrained individuals by
means of an increased maximal cardiac output (12, 59), attributable to a greater stroke volume (16, 23, 59), in addition to
greater oxygen extraction at the locomotor muscle (12, 54, 57,
59). Accordingly, we expected an increase in maximal cardiac
output during graded exercise due to a training-induced expansion of total blood volume. We originally thought that 2 wk of
HIT would increase total blood volume by means of plasma
expansion. Plasma volume responds rather rapidly in response
to high-intensity exercise training (14, 21, 22, 46), and an acute
expansion of PV in untrained individuals has shown to improve
maximal exercise capacity (33). Unlike these previous findings, however, we were unable to detect a change either PV,
TBV (Table 1), or, consequently, maximal cardiac output (Fig.
4). However, 7 wk of sprint training does not facilitate an
790
Mechanisms Explaining Exercise Improvements with HIT
•
Jacobs RA et al.
Fig. 4. Maximal cardiac output. Measures of maximal cardiac output (Qmax).
Fig. 3. Skeletal muscle oxygenation during incremental exercise. Parameters of
tissue oxygenation, specifically skeletal muscle oxygenation (⌬mStO2; A) and
deoxygenated hemoglobin and myoglobin (⌬HHbMb; B), obtained via nearinfrared spectroscopy during incremental exercise testing. Values are presented
as absolute changes from an initial value obtained prior to each respective
exercise test that was arbitrarily set equal to zero. The white and gray filled
points represent baseline and post-HIT measurements, respectively. Values
presented as means ⫾ SE. Difference between corresponding pre- and postHIT measurements: *P ⱕ 0.05, **P ⱕ 0.01. †P ⬍ 0.1 indicates a tendency for
a difference between respective baseline and post-HIT measurements.
increase in PV or BV (44) and 12 wk of both low- and
high-intensity training also failed to increase BV, PV, and
Hbmass in women (7). Thus, any acute high-intensity traininginduced expansion of PV appears to be a transient phenomenon
with a failure of initial fluid shifts to remain over time (44).
We did not expect improvements in oxygen-carrying capacity to explain improvements in maximal whole body exercise
capacity, and this assumption was substantiated by our findings
(Table 1). The rapid training-induced expansion of PV is not a
phenomenon mirrored by red blood cell volume (21, 22) or
total hemoglobin mass (22), both of which take much more
time to increase (59) if at all (61).
Two weeks of HIT has repeatedly demonstrated an ability to
increase protein expression and mitochondrial enzyme activity,
both suggestive of an enhanced oxidative capacity of the
skeletal muscle. Mitochondrial protein expression and enzyme
activity, including proteins involved in fat oxidation, TCA
Fig. 5. Peripheral fatigue resistance. The percent change (%⌬) in potentiated
quadriceps twitch force (Qtw,pot) and maximal voluntary contraction (MVC)
with time trial (TT) and maximal incremental (Max) exercise tests prior to and
again following six sessions of high-intensity interval training (HIT) are
illustrated in A and B, respectively. Values presented as means ⫾ SE.
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cycle and/or the electron transport chain (9, 10, 25, 39 – 41, 49,
63), all improve with 2 wk of HIT training. Mitochondrial
enzymatic protein expression has been reported to increase
after as few as three to five sessions of HIT with enzymes
involved in the TCA cycle and ␤-oxidation increasing before
mitochondrial respiratory complexes of the electron transport
system (49). Hitherto, these alterations in skeletal muscle
Mechanisms Explaining Exercise Improvements with HIT
Jacobs RA et al.
791
mitochondria (66). An expansion of the mitochondrial reticular
network (Fig. 2B) creates a larger oxygen sink during exercise,
reducing overall oxygen tension within a contracting myocyte
and increasing the oxygen gradient between capillaries and
mitochondria. Although not measured in the present study, a
linear association between skeletal muscle capillarization and
mitochondrial content has been established (51). Moreover,
capillary-to-fiber surface area and mitochondrial volume develop concurrently with a negligible change in capillary-tofiber surface area ratio per fiber unit mitochondrial volume
(42). Taken together with our data, this would suggest that
HIT-mediated mitochondrial expansion (Fig. 2B) might improve the diffusive capacity of the muscle. Collectively, the
larger oxygen gradient and improved diffusive capacity would
improve skeletal muscle oxygen extraction capacity during
exercise (66) as was observed (Table 2 and Fig. 3).
Resistance to the development of peripheral fatigue plays an
important role in exercise performance (13). We observed no
difference in measures of peripheral fatigue (Fig. 5, A and B)
despite decreased time to complete a set workload with a
greater mean power output (Fig. 1B) and greater peak power
output achieved (Fig. 1A). Together these data suggest that the
development of peripheral fatigue was delayed with HIT,
allowing the subjects to perform better during each respective
exercise test while maintaining the same relative level of power
output and peripheral fatigue. Accumulation of metabolic byproducts, especially inorganic phosphate (Pi), factor in the
development of peripheral fatigue (1, 68). Completing six
sessions of HIT over the course of 2 wk has been shown to
improve skeletal muscle buffer capacity (19) and reduce phosphocreatine recovery time, indirectly indicating an improved
functional oxidative capacity in skeletal muscle following
training (17, 35). Our data contribute to these previous findings
by providing direct evidence of the improvement in the oxidative capacity of the skeletal muscle with HIT (Fig. 2, A and B).
The enhanced oxidative capacities of the skeletal muscle most
likely facilitated the delayed onset of peripheral fatigue (Fig.
5), permitting improvements in overall exercise performance
(Fig. 1).
The present study could have been strengthened with the
addition of a group that engaged in more typical form of endurance training (lower intensity and longer duration), matching
work across groups. There is evidence to suggest that the transcriptional regulation of several genes involved in skeletal muscle
mitochondrial biogenesis is similar with interval and endurance
training when matching both work and time (67); however, one
primary advantage of HIT is the time efficiency of training. How
the physiological effects of a more typical endurance exercise
program would compare to those presented in this study are
unknown and require further attention.
In conclusion, we provide evidence indicating that an improved oxidative capacity of the skeletal muscle resulting from
an increase in mitochondrial content may facilitate an improvement in tissue oxygenation, delayed peripheral fatigue, and
explain the improvement in both maximal whole body exercise
capacity as well as endurance performance following 6 sessions of HIT over a 2-wk period in previously untrained adults.
GRANTS
This study was financed through grants obtained from the Swiss National
Science Foundation (320030_143745) and Team Denmark.
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protein expression have primarily served as the foundation of
mechanistic explanations for the improvements in exercise
performance with HIT (18). Measures of in vivo oxidative
capacity using phosphorus magnetic resonance spectroscopy
have also been reported to improve following 6 sessions of
HIT, further supporting these mechanistic suppositions (17,
35). While it has previously been demonstrated that the same
HIT protocol as implemented in the current study results in
increased expression and activity of COX and citrate synthase
(41), here we substantiate and expound upon these claims with
direct evidence that HIT increases skeletal muscle respiratory
capacity (Fig. 2). These improvements in skeletal muscle
respiratory capacities are most likely attributable to mitochondrial reticular expansion within the skeletal muscle as there is
primarily no difference in respiratory capacity between pre and
post HIT measurements when normalized to mitochondrial
content (Fig. 2C). Qualitative alterations in mitochondrial
function that exist across individuals that differ in aerobic
capacities (28) were not apparent after just six sessions of HIT.
Unfortunately we are unable to verify that the improvements in
exercise performance were directly facilitated by the increases
in skeletal muscle oxidative capacity and/or expansion of
skeletal muscle mitochondria as our experimental set-up does
not allow for a proper time course analysis. Future studies
could attempt to verify our findings by monitoring mitochondrial protein expression and function in parallel with measures
of exercise performance throughout a regimen of HIT. Our
results, however, do suggest that improvements in exercise
performance, oxidative capacity of the skeletal muscle and leg
oxygenation may all be attributable to an increase in skeletal
muscle mitochondria.
Leg oxygenation during exercise is also a primary determinant of maximal exercise performance (31) and may explain
measured improvements in maximal incremental performance.
Our measures of leg oxygenation, ⌬HHbMb and ⌬mStO2, can
be used as surrogate measures of oxygen extraction (6, 15). It
is generally accepted that oxygen extraction improves to some
degree with training (12, 48, 54, 56 –58), with the greatest
improvements observed in previously untrained individuals
(59). However, reported alterations in tissue oxygenation following HIT are disputed. Six sessions of HIT training reportedly improved muscle fractional oxygen extraction as measured by the changes in [HHb] kinetics with NIRS (3) whereas
negligible improvements in fractional oxygen extraction were
observed following eight sessions of HIT (43). Our results
agree with the former study (3), as we observed marked
increases in leg deoxygenation during exercise after HIT (Fig.
3) suggesting greater oxygen extraction. These results are also
in line with another study that reported greater muscle deoxygenation of the vastus lateralis muscle, suggesting improved
oxygen extraction following HIT (52). We observed that skeletal muscle deoxygenation increases at peak power output
(Table 2), at specific workloads during the graded exercise test
(Fig. 3, A and B), and throughout the time trial (Table 2). The
suggested mechanism explaining these increases in tissue deoxygenation relates back to the expansion of mitochondria
within the skeletal muscle (Fig. 2B). Oxygen extraction in the
skeletal muscle during exercise, the amount of oxygen transported per unit time between capillaries and mitochondria, is a
product of the diffusive capacity of the muscle for oxygen and
the difference between oxygen tension in the capillary and
•
792
Mechanisms Explaining Exercise Improvements with HIT
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.A.J. and C.L. conception and design of research;
R.A.J., D.F., T.C.B., S.B., P.M.C., and M.T. performed experiments; R.A.J.,
D.F., and T.C.B. analyzed data; R.A.J., D.F., and T.C.B. interpreted results of
experiments; R.A.J. prepared figures; R.A.J. drafted manuscript; R.A.J., D.F.,
T.C.B., S.B., P.M.C., and C.L. edited and revised manuscript; R.A.J., D.F.,
T.C.B., S.B., P.M.C., M.T., and C.L. approved final version of manuscript.
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