Two weeks of high-intensity aerobic interval training increases the capacity for fat oxidation during exercise in women

Abstract

Our aim was to examine the effects of seven high-intensity aerobic interval training (HIIT) sessions over 2 wk on skeletal muscle fuel content, mitochondrial enzyme activities, fatty acid transport proteins, peak O(2) consumption (Vo(2 peak)), and whole body metabolic, hormonal, and cardiovascular responses to exercise. Eight women (22.1 +/- 0.2 yr old, 65.0 +/- 2.2 kg body wt, 2.36 +/- 0.24 l/min Vo(2 peak)) performed a Vo(2 peak) test and a 60-min cycling trial at approximately 60% Vo(2 peak) before and after training. Each session consisted of ten 4-min bouts at approximately 90% Vo(2 peak) with 2 min of rest between intervals. Training increased Vo(2 peak) by 13%. After HIIT, plasma epinephrine and heart rate were lower during the final 30 min of the 60-min cycling trial at approximately 60% pretraining Vo(2 peak). Exercise whole body fat oxidation increased by 36% (from 15.0 +/- 2.4 to 20.4 +/- 2.5 g) after HIIT. Resting muscle glycogen and triacylglycerol contents were unaffected by HIIT, but net glycogen use was reduced during the posttraining 60-min cycling trial. HIIT significantly increased muscle mitochondrial beta-hydroxyacyl-CoA dehydrogenase (15.44 +/- 1.57 and 20.35 +/- 1.40 mmol.min(-1).kg wet mass(-1) before and after training, respectively) and citrate synthase (24.45 +/- 1.89 and 29.31 +/- 1.64 mmol.min(-1).kg wet mass(-1) before and after training, respectively) maximal activities by 32% and 20%, while cytoplasmic hormone-sensitive lipase protein content was not significantly increased. Total muscle plasma membrane fatty acid-binding protein content increased significantly (25%), whereas fatty acid translocase/CD36 content was unaffected after HIIT. In summary, seven sessions of HIIT over 2 wk induced marked increases in whole body and skeletal muscle capacity for fatty acid oxidation during exercise in moderately active women.

Full text

TWO WEEKS OF HIGH-INTENSITY AEROBIC INTERVAL TRAINING INCREASES
THE CAPACITY FOR FAT OXIDATION DURING EXERCISE IN WOMEN
Jason L. Talanian1, Stuart D.R. Galloway2, George J. F. Heigenhauser3, Arend Bonen1 &
Lawrence L. Spriet1
1Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario,
Canada. 2Department of Sport Studies, University of Stirling, Scotland.
3Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Running Title: Fat metabolism during high intensity interval training
Corresponding author: Jason L. Talanian
Department of Human Health and Nutritional Sciences
University of Guelph
Guelph, Ontario
Canada, N1G 2W1
Tel: 1-519-824-4120 x53907
Fax: 1-519-763-5902
Email: jtalania@uoguelph.ca
Page 1 of 38 Articles in PresS. J Appl Physiol (December 14, 2006). doi:10.1152/japplphysiol.01098.2006
Copyright © 2006 by the American Physiological Society.

2
ABSTRACT
Our aim was to examine the effects of seven high intensity aerobic interval training (HIIT)
sessions over two weeks on skeletal muscle fuel content, mitochondrial enzyme activities, fatty
acid transport proteins, VO2peak, and whole body metabolic, hormonal and cardiovascular
responses to exercise. Eight females participated in the study (22.1 ± 0.2 yrs, 65.0 ± 2.2 kg,
VO2peak: 2.36 ± 0.24 lmin-1). Subjects performed a VO2peak test and a 60-min cycling trial at
~60% VO2peak prior to and following training. Each session consisted of ten, 4-min bouts at
~90% VO2peak with 2-min rest between intervals. Training increased VO2peak by 13%.
Following HIIT, plasma epinephrine and heart rate were lower during the final 30-min of the 60-
min cycling trial at ~60% pre-training VO2peak. Exercise whole body fat oxidation (PRE: 15.0 ±
2.4, POST: 20.4 ± 2.5 g) increased 36% following HIIT. Resting muscle glycogen and
triacylglycerol contents were unaffected by HIIT, but net glycogen use was reduced during the
post-training 60-min cycling trial. HIIT significantly increased muscle mitochondrial -HAD
(PRE: 15.44 ± 1.57, POST: 20.35 ± 1.40 mmol
min-1
kg wm-1) and citrate synthase (PRE: 24.45
± 1.89, POST: 29.31 ± 1.64 mmol
min-1
kg wm-1) maximal activities by 32% and 20%, while
cytoplasmic HSL protein content was not significantly increased. In addition, total muscle
FABPpm content increased significantly (25%), while FAT/CD36 content was unaffected
following training. In summary, seven sessions of HIIT over two weeks induced marked
increases in whole body and skeletal muscle capacity for fatty acid oxidation during exercise in
moderately active women.
Key words: fatty acid metabolism, mitochondrial enzymes, aerobic capacity, fatty acid transport
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INTRODUCTION
Endurance exercise training results in an improved capacity for whole body fat oxidation
that is associated with increased mitochondrial volume as assessed via increases in citrate
synthase and -hydroxy-acyl-CoA dehydrogenase (-HAD) activities (19, 30, 37, 52) . These
along with other adaptations not only improve the potential for muscle to utilize lipids as a
substrate for energy, but are also associated with improved insulin sensitivity (20) and health.
Improving skeletal muscle fatty acid oxidation is of considerable importance for individuals
attempting to increase fat oxidation during exercise and also for athletes attempting to spare
carbohydrate during competition.
It has commonly been observed that 6-12 weeks of exercise training at a moderate
intensity (MIT, 60-75% VO2peak) can improve aerobic capacity and maximal mitochondrial
enzyme activities (19, 28, 29, 37). In addition, sprint interval training (SIT) at very high power
outputs (150-300% VO2peak power) for 6-7 weeks produces similar results (41, 48, 55). Recent
evidence has also shown that daily sessions of MIT (two hours/day) for only 6-10 days can
improve aerobic capacity and mitochondrial enzyme activities (10, 52), although not all short-
term MIT protocols have reported similar increases (46, 47). Even as little as six SIT sessions in
two weeks has also been shown to increase citrate synthase activity but without an increase in
VO2peak (5). Both the MIT and SIT short duration (2 wk) protocols produce substantial training
effects and health benefits in a brief period of time. However, MIT for two hours a day is time
consuming and difficult to complete and SIT is performed at an all-out maximal intensity that is
very challenging and may be too intense to sustain for people beginning a training program.
Two weeks of high intensity aerobic interval training (HIIT), performed at an exercise
intensity (80-95% VO2peak) between moderate and sprint training paradigms, may offer similar
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benefits to MIT and SIT. Training studies utilizing HIIT over a longer period of time (4-6 wk)
have reported increases in high intensity exercise performance, muscle buffering capacity, whole
body exercise fat oxidation rates and aerobic capacity (15, 39, 63). However, no studies have
examined whether aerobic capacity and skeletal muscle metabolic adaptations are improved in as
little as two weeks of HIIT.
Our aim was to investigate the effect of seven HIIT sessions over a two week period on
skeletal muscle metabolism during a 60 min steady state cycling trial in recreationally active
women. We measured aerobic capacity, exercise whole body fat oxidation, and muscle glycogen
and triacylglycerol (TG) contents, maximal mitochondrial enzyme activities, and fatty acid
transport proteins prior to and following training. In addition, we also evaluated the effects of
training on circulatory substrates and on respiratory responses during HIIT throughout the
second and seventh training session.
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METHODS
Eight healthy recreationally active females (22 ± 1 years, 65.0 ± 2.2 kg; VO2peak: 2.36 ±
0.24 lmin-1) volunteered to participate in the study. On average, subjects engaged in recreational
physical activity 2-3 days a week. Most subjects did not limit their exercise to one type, but
common activities included weight lifting, soccer, cycling, swimming and walking. Subjects
were fully informed of the purpose of the study and of potential risks before giving written
consent. This study was approved by the Ethics Committees at McMaster University and the
University of Guelph.
Preliminary testing. Prior to the study subjects reported to the laboratory on two occasions. On
the first visit, subjects performed an incremental cycling (Lode Excalibur, Quinton Instrument,
Netherlands) test to exhaustion to determine VO2peak. Respiratory gases were collected and
analyzed using a metabolic cart (Sensormedic, Vmax 229, Yorba Linda, CA). The second visit
was to verify appropriate power outputs for the experimental trials. Subjects cycled for 15 min at
60% VO2peak to establish the power output for the 60 min trial. They then performed 4-6 bouts
of cycling at 90% VO2peak, with each bout lasting 4 min and separated by 2 min of rest to
establish power outputs for the HIIT sessions. Following two weeks (7 sessions) of HIIT,
subjects repeated the incremental cycling test to exhaustion to establish the post-training
VO2peak.
Cycle trials at ~60% VO2peak. Subjects performed a 60 min cycling trial at a moderate intensity
(~60% VO2peak) before and three days following seven sessions of HIIT. Subjects arrived at the
laboratory 3-4 hours post-prandial. They abstained from strenuous exercise and recorded their
diet in the 24 hours prior to the trial. Three to four hours prior to the 60 min ride, subjects
received a meal that was provided for them. Prior to the post-training 60 min ride subjects
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replicated the same diet they ingested before the pre-training ride. A Teflon catheter was inserted
into an antecubital vein for blood sampling and the catheter was kept patent by flushing with
0.9% saline. One leg was prepared for percutaneuos needle biopsy sampling of the vastus
lateralis muscle. Three incisions were made in the skin and deep fascia under local anesthesia
(2% xylocaine without epinephrine) for three separate biopsies. Immediately prior to exercise,
venous blood (5 ml) and one muscle biopsy were obtained while the subject rested on a bed. All
muscle samples were immediately frozen in liquid nitrogen for subsequent analysis. Subjects
then cycled for 60 min at ~60% VO2peak at a constant cadence (78-85 rpm) on the Lode
ergometer. Respiratory gases were collected between 13-17, 28-32, 43-47 and 55-59 min of
exercise for the measurements of VO2 and VCO2 and the calculation of the respiratory exchange
ratio (RER). These parameters were used to calculate whole body fat and carbohydrate oxidation
using the non-protein RER table (16) and according to the following equations: carbohydrate
oxidation = 4.585 (VCO2) – 3.226 (VO2) and fat oxidation = 1.695 (VO2) – 1.701 (VCO2) (45).
Venous blood samples were obtained at 15, 30, 45 and 60 min of exercise. Immediately
following exercise, two muscle biopsies were taken with the subject sitting on the cycle
ergometer. The same procedure was repeated following HIIT with muscle biopsies taken from
the other leg.
High intensity interval training (HIIT). Two days following the initial 60 min trial subjects began
training every other day completing seven HIIT sessions in 13 days (Fig. 1). All training sessions
were supervised. Each session consisted of ten, 4 min cycling bouts at 90% VO2peak separated
by 2 min of rest. Heart rate (HR) was recorded throughout training and was held constant at
~90% of HRmax by increasing the power output as training progressed. Required adjustments in
training power output were made at the beginning of sessions and all subjects experienced three
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power output increases during the initial six training sessions. During the seventh training
session subjects cycled at the same power output as the second training session to make training
related comparisons. During training sessions 2 and 7, respiratory gases and venous blood
samples (Teflon catheter) were collected prior to and immediately following bouts 1, 3, 5, and
10. Throughout the two weeks of training, subjects maintained their recreational activities they
were engaged in prior to training.
Analyses.
Blood measurements. Venous blood was collected in sodium-heparin tubes. A portion (1.5 ml)
was added to 30 µl of EGTA and reduced glutathione, centrifuged (10,000 x g for 3 min) and the
supernatant was analyzed for epinephrine using an enzymatic immunoassay (Labor Diagnostika
Nord, Nordhorn, Germany). A second portion (200 µl) was added to 1 ml of 0.6M perchloric
acid, centrifuged and the supernatant was analyzed for blood glucose, lactate and glycerol using
fluorometric techniques (1). A third portion (1.5 ml) was centrifuged and the plasma was
analyzed for free fatty acids (FFA) using an enzymatic colorimetric technique (Wako NEFA C
test kit, Wako Chemicals, Richmond, VA).
Muscle enzyme activities. Resting frozen wet muscle samples (~6-10 mg) were homogenized in
0.1 M KH2PO4 and BSA, freeze-thawed three times and the maximal activities of citrate
synthase and -HAD were determined on a spectrophotometer (at 37oC) using methods formerly
described (53). The muscle homogenate was analyzed for total creatine (Cr) and enzyme
measurements were normalized to the highest total pre/post Cr measured among each subject.
Muscle metabolites. A portion of the resting and first post exercise muscle biopsy were freeze
dried, powdered and dissected free of visible connective tissue, fat and blood. One aliquot of
freeze dried powdered muscle (~10 mg) was extracted in 0.5 M HClO4/l mM EDTA and
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neutralized with 2.2 M KHCO3. The supernatant was used to measure Cr, phosphocreatine (PCr),
ATP and lactate. A second aliquot (2-4 mg) was extracted in 0.1 M NaOH, neutralized with 0.1
M HCl/0.2 M citric acid/0.2 M Na2PO4 and amyloglucosidase was added to breakdown glycogen
to glucose which was measured spectrophotometrically (1). The Folch extraction was used on a
third freeze dried aliquot (6-9 mg) to separate TG from the muscle (17). The TG were degraded
and the resultant glycerol was extracted for the determination of IMTG content (1). The total Cr
content of freeze dried muscle samples was similar pre- and post-training and therefore all freeze
dried measurements were normalized to the highest total Cr measured among all six biopsies
from each subject.
Western blots. Frozen wet muscle samples (50-70 mg) from the second post-exercise biopsy
were initially homogenized in a buffer containing 210 mM sucrose, 2 mM EGTA, 40 mM NaCl,
30 mM HEPES, 20mM EDTA, PMSF and DMSO. A second buffer containing 1.17 M KCl and
58.3 M tetra-sodium pyrophosphate was added, samples were centrifuged (50,000 rpm for 75
min) and the supernatant was discarded. Samples were then homogenized in a third buffer (10
mM tris-base/1 mM EDTA), 16% SDS was added, samples were centrifuged (3000 rpm for 15
min) and the supernatant was used to determine fatty acid translocase (FAT/CD36), plasma
membrane fatty acid binding protein (FABPpm) and hormone sensitive lipase (HSL) total content
through a western blot technique. Briefly, samples were separated on an 8% SDS-
polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. A monoclonal
antibody (MO25) was used to specifically detect FAT/CD36 content (42), a FABPpm/mAspAT
polyclonal antibody was used was to determine FABPpm content (6) and a polyclonal antibody
for HSL (ProSci, Poway, CA) was used to determine total HSL content.
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Statistics. All data are presented as means ± SE. FABPpm, FAT/CD36, and HSL were analyzed
using paired t-tests. All other data were analyzed by two way repeated measures ANOVA (time
x trial) to determine significant differences during the 60 min trials and between training sessions
2 and 7. Specific differences were identified using a student Newman-Keuls post hoc test.
Statistical significance was accepted at a level of p < 0.05.
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RESULTS
High Intensity Interval Training.
VO2peak increased from 2.36 ± 0.24 l
min-1 (36.3 ± 3.7 ml
kg-1
min-1) prior to training to
2.66 ± 0.21 l
min-1 (40.9 ± 3.2 ml
kg-1
min-1) (13%) following seven HIIT sessions consisting of
ten 4 min bouts at ~90% VO2peak with 2 min rest between bouts. Initial training power outputs
(163-227 W) were increased by an average of 19.0 ± 0.6 W from the first to the sixth training
session to maintain a constant HR during the exercise sessions. For comparison, training power
outputs during session 7 were reduced to match the same absolute power outputs as session 2.
The average absolute VO2 during training sessions 2 and 7 were not different (Table 1). During
training session 2, VO2 reached 86% of pre-training VO2peak in bout 1 and 95% of pre-training
VO2peak in bout 10, while the same power outputs represented 77% and 84% of the post-
training VO2peak in bouts 1 and 10 of training session 7 (Table 1). The peak HR attained during
session 2 ranged from 171 ± 2 beats
min-1 during bout 1 to 181 ± 1 beats
min-1 during bout 10
and was significantly lower throughout training session 7 (Table 1).
Venous plasma epinephrine concentrations increased during each cycling bout during
training session 2 and reached the highest level following bout 10 (Fig. 2). There was a
significantly blunted epinephrine response in session 7 following bouts 3 and 10. Whole blood
lactate concentrations in session 2 increased following bouts 1 and 3 and reached a plateau
during the remaining bouts (Table 1). There was no difference in the lactate response to cycling
during session 7. Whole blood glucose and plasma FFA concentrations were unchanged
throughout the training bouts in both training sessions (Table 1). Whole blood glycerol
concentrations increased during the training bouts in both sessions 2 and 7, but were significantly
lower prior to and following bout 10 in session 7 (Table 1).
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Cycling at ~60% VO2peak Pre- and Post-Training.
Subjects cycled for 60 min at 101.3 ± 4.2 W prior to and following HIIT. This power
output represented 63.9 ± 2.6 % of the pre-training VO2peak and 55.2 ± 2.2 % of the post-
training VO2peak (Table 2). The RER was significantly lower following HIIT (Table 2) and the
estimated whole body fat oxidation was significantly higher at 30, 45 and 60 min of cycling (Fig.
2). Total fat oxidation during the 60 min trial prior to training was 15.0 ± 2.4 g and increased
36% following HIIT to 20.4 ± 2.5 g. There was a reciprocal decrease in whole body
carbohydrate oxidation at 30, 45 and 60 min following training (Fig. 3). Total carbohydrate
oxidation prior to training was 80.7 ± 2.2 g and decreased 23% following HIIT to 62.1 ± 1.4 g.
HR was significantly lower following training at 45 and 60 min of cycling (Fig. 4).
Plasma epinephrine concentrations increased during exercise in both trials, but were blunted at
30 and 60 min of exercise post-training (Fig. 4). Plasma lactate was significantly increased above
rest at all exercise time points in both trials but the increase was blunted following HIIT at 15, 30
and 45 min of exercise (Fig. 4).
Plasma FFA decreased from rest at 15 min and then increased over time and was
significantly higher than rest following 60 min of exercise in the pre-training trial (Table 3).
Post-training, plasma FFA was not altered from rest for 45 min but increased significantly above
rest at 60 min. Whole blood glycerol was elevated above rest at all exercise time points in both
trials and was significantly higher post- vs. pre-training at 30 and 60 min of exercise (Table 3).
Blood glucose was unchanged by exercise in both trials, but was higher at rest, 45 and 60 min of
exercise following training (Table 3).
Muscle Analysis
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Maximal -HAD activity (PRE: 15.44 ± 1.57, POST: 20.35 ± 1.40 mmol
min-1
kg wm-1)
increased by 32% and maximal citrate synthase activity (PRE: 24.45 ± 1.89, POST: 29.31 ± 1.64
mmol
min-1
kg wm-1) increased by 20% following HIIT (Fig. 5). There was a non-significant
increase in muscle HSL protein content (~13%) following training (Fig. 6). Total muscle
FABPpm content increased by 25% following training, while muscle FAT/CD36 protein content
was unchanged (Fig. 6).
Resting muscle glycogen content was unaffected by training, but net muscle glycogen
utilization was decreased by 12% following 60 min of exercise post-training (Table 4). IMTG
content decreased 12% and 17% following 60 min of cycling pre- and post-training respectively,
but there was no difference between the trials (Table 4).
Resting muscle PCr was similar in both trials, but PCr was higher at 60 min following
training, such that net PCr degradation was significantly decreased by 40% following HIIT.
Muscle ATP was unchanged by exercise following both trials, but post-training ATP contents
were lower than pre-training at 60 min (Table 4). Muscle free ADP at 60 min was lower
following the post-training trial (Table 4). Muscle lactate contents increased from rest following
60 min of exercise to the same extent in both trials (Table 4).
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DISCUSSION
This study examined the effects of two weeks of high-intensity interval training (HIIT) at
~90% VO2peak on whole body and muscle metabolic responses to exercise at ~60% pre-training
VO2peak in recreationally active females. This is the first study using this short duration HIIT
protocol to measure both whole body responses and metabolic adaptations in skeletal muscle.
Training resulted in increased VO2peak, whole body fat oxidation during exercise and maximal
mitochondrial enzyme activities (citrate synthase, -HAD) following only seven HIIT sessions in
two weeks. Training also increased the skeletal muscle content of the fatty acid transport protein
FABPpm, which may have contributed to the observed increases in whole body fat oxidation.
Training Induced Increases in VO2peak and Muscle Mitochondrial Enzymes
Classic responses to the traditional long duration (> 24 hr accumulated training)
submaximal training protocols are an improved aerobic capacity (9, 19, 28), increased whole
body fat oxidation, and increases in skeletal muscle mitochondrial enzyme activities (28, 29, 37).
It has also been shown that as little as 6-7 two hour sessions at ~60-70% VO2peak increases
aerobic capacity, whole body fat oxidation and mitochondrial enzyme activities (10, 52). While a
lack of a control group training for a similar duration at a lower cycling intensity limits our
interpretations of our results, we are confident that previous literature on short term endurance
training reveals the significance of our short term HIIT protocol.
Long duration (6-7 wk) intermittent sprint protocols have also produced significant
improvements in VO2peak and mitochondrial enzyme activity (25, 41, 48, 55). Moreover, there
has also been recent interest into the adaptive responses of as little as six sprint training sessions
over two weeks (~15-18 min of training) (4, 5, 18). These studies reported significant increases
in exercise performance and skeletal muscle citrate synthase activity and cytochrome C oxidase
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protein content, without increases in VO2peak or -HAD activity. The uniqueness of the present
study is that a training intensity (~90% VO2peak) that is intermediate between classic
submaximal and sprint training paradigms resulted in increases in VO2peak, skeletal muscle
citrate synthase and -HAD activity, and whole body fat oxidation. Even though in both the
present study and the short duration sprint studies subjects trained for only two weeks, the total
training time was ~4.7 hr in our study vs. 15-18 min of training in the sprint studies. This argues
that with interval training, there is a specific amount of exercise that is required for VO2peak to
increase.
Our HIIT protocol as well as others training for two hours a day at ~60-70% VO2peak
(52) observed similar increases in -HAD activity following only seven training sessions. In
contrast, other 2 hour/day protocols lasting 5-7 days (46, 47) and six sprint (5) training sessions
did not observe significant increases in -HAD. The data from our high intensity intermittent
training protocol suggests that a combination of high training intensities, the duration of each
bout (4 min), and several rest to exercise transitions provides a powerful stimulus for increasing
the enzyme contents of many of the metabolic pathways in the mitochondria in a short period of
time. It is not clear why the 90% VO2peak intermittent training protocol increases both citrate
synthase and -HAD activity, but HIIT offers a mechanism to quickly increase muscle
mitochondrial capacity as well as whole body fat oxidation and VO2peak in untrained
individuals.
Training Induced Metabolic Responses to 60 min of Cycling at ~60% Pre-Training VO2peak
Whole Body Fat Oxidation. In the present study, seven intermittent HIIT sessions at ~ 90%
VO2peak increased post-training whole body fat oxidation during 60 min of cycling at ~60% of
pre-training VO2peak. This is a classic response typically observed with longer duration
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endurance training studies (27, 34, 49), but the present adaptations in whole body fat oxidation
occurred with only two weeks of training. Previously, incorporation of interval training into
cyclists’ exercise regime yielded similar results. Well-trained cyclists replaced a portion (~15%)
of their normal training with six weeks of HIIT bouts at ~80% of VO2peak resulting in an
enhanced whole body fat oxidation during exercise (62). Therefore, HIIT offers a short duration
stimuli for elite endurance athletes to increase fat oxidation during exercise above an already
high endurance training-induced level of fatty acid oxidation.
Reduced Glycogenolysis. Muscle glycogenolysis was reduced by 12% during 60 min of cycling
post-training. Muscle glycogen phosphorylase, a key regulatory enzyme in glycogenolysis, is
activated by epinephrine via the cyclic AMP second messenger system and the release of
calcium during contractions. The activity of phosphorylase in the active “a” form is also
stimulated via the contraction-induced accumulation of allosteric regulators, free ADP and AMP.
The blunted plasma epinephrine response and reduced accumulations of free ADP and AMP in
the present study are classic training-induced alterations in traditional moderate intensity
endurance protocols (21, 36, 46). These changes were consistent with the decreased glycogen use
that occurred during the 60 min cycling trial in the present study. Once again, the uniqueness of
the present work is that the classic training-induced shifts in fuel use during exercise were
present following as little as seven HIIT sessions over two weeks.
Unlike most training studies where resting muscle glycogen content increased following
training (5, 10, 22, 48), resting glycogen content was unchanged in the present study. It appears
that the present training stimulus (glycogen degradation each training day) and number of
training days did not appear to be sufficient to increase resting muscle glycogen.
Skeletal Muscle Fat Metabolism
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Increases in skeletal muscle fat oxidation likely result from a number of adaptations,
including an increase in mitochondrial volume (30) and alterations at several regulatory steps;
adipose tissue lipolysis of triglycerides (TG) to fatty acids (60), transport of fatty acids into the
cell, intramuscular lipolysis of TG to fatty acids, and ultimately fatty acid transport into the
mitochondria (2, 3). Exercise training results in a greater contribution of energy being derived
from fatty acids that is stored in peripheral adipose tissue and IMTG stores (34, 58, 59). It has
also been shown that exercise trained individuals use more intramuscular TG as an energy source
than untrained individuals (34, 50). However, in the present study, training did not result in a
significant increase (35%) in IMTG utilization (PRE: 5.4 ± 3.5 vs. POST: 7.3 ± 3.7 mmol
kg DM
-1), but 60 min of exercise may have been too short to detect a training effect.
In the present study we did not see a significant increase in HSL protein content
following HIIT. HSL is believed to be key regulatory enzyme in lipolysis of IMTG stores (33,
61). However, it may be that our training protocol was not long enough to stimulate significant
adaptations in skeletal muscle HSL content and further studies are warranted to assess if
adaptations increase further or plateau following longer HIIT training protocols.
A third regulatory step that may limit skeletal muscle fat oxidation is through the
transport of fatty acids across the plasma and mitochondrial membranes. Although previously
viewed as a completely passive process (24), evidence now suggests that LCFA membrane
transport is a highly regulated process involving several transporters (3, 40). We measured two
transport proteins of interest, FABPpm and FAT/CD36. Training resulted in a significant increase
in total FABPpm content, but no change in FAT/CD36 content. FABPpm has been identified as a
plasma membrane LCFA transport proteins and inhibition of this transporter decreases LCFA
uptake (56). Three weeks of long duration (15 training sessions lasting 1-2 hr per session) knee
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17
extension exercise resulted in an increased whole muscle FABPpm content (38), but the present
study is the first to demonstrate an increase in FABPpm content using HIIT over only seven
training sessions.
The absence of an increase in FAT/CD36 content does not necessarily imply that there
was no increase in the transport potential of LCFA through FAT/CD36. Research suggests that
FAT/CD36 is located at the plasma membrane (3), within the intracellular fraction and on the
mitochondrial membrane (2, 32), with FAT/CD36 content on the mitochondria following a
similar trend to oxidative capacity within tissue types (heart > red muscle > white muscle) in
rodents (7). Therefore, it remains possible that there was a shift in the fractional concentrations
of FAT/CD36 on the mitochondria and plasma membrane that could increase LCFA uptake (2, 7,
32)
Female Exercise Training Studies
Similar to men, well-trained women have enhanced aerobic and mitochondrial enzyme
capacities compared to women less trained (12, 13). As well, traditional endurance training
studies using mixed male and female populations have shown that training increases these
markers of fitness as well as increasing whole body fat oxidation (11, 31, 44, 51). However, the
present study is the first to use an interval training protocol at ~90% VO2peak using exclusively
female subjects to observe increases in mitochondrial enzyme activities, VO2peak and whole
body fat oxidation. There have been varying results showing that women utilize slightly different
proportions of carbohydrate and fat sources for fuel than men (43, 57) and others that have
observed no gender differences (8). As well, some studies have shown that substrate utilization
varies during different phases of the menstrual cycle (23, 64) and others have shown no
difference in substrate utilization between varying menstrual cycle phases (26, 35, 54). In this
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study, our whole body fat oxidation rates following HIIT were very convincing with all eight
subjects using a higher absolute rate and a greater percentage for energy than prior to training.
Future studies are necessary to compare genders following HIIT.
In summary, seven sessions of HIIT training over a two week period offers a short
duration stimulus to improve whole body fat oxidation and the capacity for skeletal muscle to
oxidize fat. HIIT is a realistic type of exercise that can be performed by elite athlete as well as
untrained individuals. Our protocol along with other HIIT and SIT protocols reveal “the potency
of exercise intensity for stimulating adaptations in skeletal muscle that improve performance and
have implications for improving health” (14). The short duration of our training provides a tool
that can be incorporated into existing training protocols to maximize training adaptations in a
short period of time, or can be used by untrained individuals to improve initial fitness with only
three hours of training a week for two weeks.
Acknowledgements:
The authors thank Lindsay Crabbe and Erin Weersink for excellent technical assistance. This
study was supported by operating grants from the Canadian Institutes of Health Research
(L.L.S., G.J.F.H. and A.B.), a Gatorade Sports Science Institute Award (J.L.T.), the Natural
Science and Engineering Research Council of Canada (L.L.S and A.B.), the Physiological
Society and Carnegie Trust for the Universities of Scotland (S.D.R.G.) and the Canada Research
Chair Program (A.B.). A. Bonen is the Canada Research Chair in Metabolism and Health.
Page 18 of 38

19
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27
Table 1.
Respiratory, heart rate and venous blood measurements during high intensity interval training sessions 2 and 7.
0 min 4 min 0 min 4 min 0 min 4 min 0 min 4 min
VO
2
(L
•
min
-1
)
Session 2 - 2.03 ± 0.17 - 2.19 ± 0.16 - 2.19 ± 0.17 - 2.24 ± 0.15
Session 7 - 2.07 ± 0.17 - 2.17 ± 0.19 - 2.21 ± 0.26 - 2.25 ± 0.20
% VO
2
peak
S2 (% of Pre-training VO
2
peak) -
86.1 ± 2.5
-92.8 ± 2.5
†
-92.9 ± 2.5
†
-94.8 ± 2.3
†
S7 ( % of Post-training VO
2
peak) -77.7 ± 2.3
*
-81.5 ± 2.6
†*
-83.0 ± 3.5
†*
-84.4 ± 2.6
†*
Heart Rate (beats
•
min
-1
)
Session 2 72 ± 2 171 ± 2 125 ± 4
†
180 ± 2
†
133 ± 3
†
181 ± 2
†
132 ± 3
†
181 ± 1
†
Session 7 69 ± 4 167 ± 2 125 ± 3
†
174 ± 2
†*
129 ± 3
†
177 ± 2
†*
126 ± 4
†
177 ± 2
†*
Lactate (mM)
Session 2 0.6 ± 0.1 2.1 ± 0.3 3.1 ± 0.2
†
3.4 ± 0.2
†
3.5 ± 0.1
†
3.6 ± 0.2
†
3.2 ± 0.2
†
3.3 ± 0.2
†
Session 7 0.6 ± 0.1 2.0 ± 0.2 2.8 ± 0.1
†
3.0 ± 0.2
†
2.9 ± 0.1
†
3.3 ± 0.2
†
3.3 ± 0.2
†
3.5 ± 0.2
†
Glucose (mM)
Session 2 4.84 ± 0.12 4.58 ± 0.25 4.90 ± 0.23 4.53 ± 0.21 5.16 ± 0.32 4.94 ± 0.17 5.34 ± 0.19 4.51 ± 0.18
Session 7 4.91 ± 0.14 4.68 ± 0.10 4.46 ± 0.19 4.11 ± 0.17 4.80 ± 0.20 4.58 ± 50.21 5.34 ± 0.28 4.77 ± 0.24
FFA (mM)
Session 2 0.44 ± 0.10 0.27 ± 0.04 0.34 ± 0.06 0.26 ± 0.03 0.43 ± 0.11 0.28 ± 0.04 0.59 ± 0.14
†
0.38 ± 0.11
Session 7 0.32 ± 0.06 0.27 ± 0.04 0.28 ± 0.03 0.22 ± 0.03 0.31 ± 0.03 0.27 ± 0.03 0.42 ± 0.04 0.27 ± 0.03
Glycerol (µM)
Session 2
35.4 ± 6.3
41.6 ± 7.6 59.2 ± 8.0
†
67.0 ± 7.5
†
87.2 ± 11.9
†
84.2 ± 10.4
†
109.4 ± 12.0
†
127.7 ± 13.5
†
Session 7
45.8 ± 4.6
55.9 ± 3.8 64.0 ± 4.6
†
65.2 ± 6.0
†
74.7 ± 4.6
†
79.2 ± 5.6
†
88.6 ± 3.9
†*
97.6 ± 5.9
†*
Values are means ± SE, n = 8. S2, Session 2; S7, Session 7; FFA, free fatty acids. † Significantly higher (p < 0.05) than the same time point during bout 1.
* Significantly lower then the same time point during Session 2
Bout 10Bout 1 Bout 3 Bout 5
Page 27 of 38

28
Table 2. Effects of high intensity interval training on VO
2
and respiratory exchange ratio during 60 min of cycling
at ~60% pre-training VO
2
peak.
15 min 30 min 45 min 60 min
VO
2
(l
•
min
-1
)
Pre 1.47 ± 0.06 1.50 ± 0.06 1.51 ± 0.05 1.51 ±0.05
Post 1.46 ± 0.06 1.45 ± 0.06 1.44 ± 0.05 1.45 ±0.06
% VO
2
peak
Pre (% of Pre-training VO
2
peak)
62.1 ± 2.6
64.1 ± 2.9
64.6 ± 2.3
64.8 ± 3.1
Post (% of Post-training VO
2
peak)
55.5 ± 2.3
*
55.4 ± 2.6
*
54.9± 2.2
*
55.2 ± 2.9
*
RER
Pre 0.92 ± 0.02 0.91 ± 0.02 0.88 ± 0.01
*
0.88 ±0.02
Post 0.89 ± 0.02 0.85 ± 0.02
†*
0.84 ± 0.02
†*
0.84 ±0.02
†*
Values are means ± SE, n = 8. VO
2
, oxygen consumption; RER, respiratory exchange ratio; Pre, pre-training; Post, post-training.
† Significantly different (p < 0.05) from 15 min of the same trial. * Significantly different than the same time point during the pre-training trial.
Page 28 of 38

29
Table 3.
Effects of high intensity interval training on venous blood measurements during 60 min of
cycling at ~60% pre-training VO
2
peak.
0 min 15 min 30 min 45 min 60 min
FFA (mM)
Pre 0.60 ± 0.14 0.39 ± 0.06
†
0.49 ± 0.10
0.65 ± 0.14
0.87 ± 0.16
†
Post 0.52 ± 0.11 0.42 ± 0.06 0.48 ± 0.06 0.56 ± 0.14 0.72 ± 0.16
†
Glycerol (µM)
Pre
60.0 ± 5.6
67.2 ± 5.1
†
89.6 ± 5.1
†
118.9 ± 7.1
†
140.5 ± 10.5
†
Post 54.4 ± 3.8 79.2 ± 3.8
†
110.0 ± 12.6
†*
131.3 ± 12.8
†
166.4 ± 13.6
†*
Glucose (mM)
Pre 4.4 ± 0.2 4.8 ± 0.3 4.7 ± 0.3 4.5 ± 0.3 4.4 ± 0.3
Post 5.2 ± 0.2
*
4.8 ± 0.3
5.1 ± 0.4
5.2 ± 0.3
*
5.2 ± 0.4
*
Values are means ± SE, n = 8. Pre, pre-training; Post, post-training; FFA, free fatty acid. † Significantly different (p < 0.05)
from 0 min of the same trial.
*
Significantly different than the same time point during the Pre trial.
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30
Table 4. Effects of high intensity interval training on skeletal muscle measurements during 60 min of cycling at ~60% pre-training VO
2
peak.
0 min 60 min 0 min 60 min Pre Post
Glycogen 468.6 ± 25.0 136.5 ± 17.4
†
474.6 ± 25.8 182.4 ± 15.5
†*
332.1 ± 19.1 292.2 ± 22.3
*
IMTG
46.4 ± 2.6
41.0 ± 3.0
43.1 ± 3.3
35.8 ± 3.4
5.4 ± 3.5 7.3 ± 3.7
Phosphocreatine
76.9 ± 3.3
53.5 ± 4.3
†
77.2 ± 3.2 63.1 ± 3.3
†
23.4 ± 5.8 14.1 ± 3.3
*
ATP
24.1 ± 1.2
24.2 ± 1.6
22.4 ± 0.8 21.5 ± 0.8
*
-0.1 ± 1.1 0.9 ± 0.3
ADPf 101.8 ± 10.5 198.0 ± 41.5
†
88.1 ± 2.3 120.1 ± 9.7
†*
96.1 ± 39.9 32.1 ± 9.7
*
AMP
f
0.46 ± 0.12 1.87 ± 0.81
†
0.33 ± 0.01 0.66 ± 0.08
†
1.41 ± 0.8 0.33 ± 0.09
*
Lactate 3.9 ± 0.4 11.1 ± 0.9
†
3.7 ± 0.5 9.9 ± 0.8
†
7.2 ± 1.0 6.2 ± 1.6
Values are means ± SE, n = 8. IMTG, intramuscular triacylglycerol; ADPf, free adenosine diphosphate; AMPf, free adenosine monophosphate.
Data are mmol
•
kg dry mass-1 except for ADP and AMP (µmol
•
kg dry mass-1). † Significantly different (p < 0.05) from 0 min of the same trial.
* Significantly different than the same time point Pre-training
Pre-training Post-training
Page 30 of 38

31
FIGURE 1. High intensity interval training study design. HIIT, High intensity interval
training; HIIT; S#, training session #.
FIGURE 2. Venous plasma epinephrine concentrations during high intensity interval
training sessions 2 and 7. Values are mean ± SE, n = 8. * Significantly lower than the
same time point during session 2 (p < 0.05).
FIGURE 3. Effects of high intensity interval training on whole body fat and
carbohydrate oxidation measurements during 60 min of cycling at ~60% pre-training
VO2peak. Values are means ± SE, n = 8. * Significantly different than the same time point
pre training (p < 0.05).
FIGURE 4. Effects of high intensity interval training on heart rate, venous plasma
epinephrine and whole blood lactate concentrations during 60 min of cycling at ~60%
pre-training VO2peak. Values are mean ± SE, n = 8. * Significantly lower than the same
time point pre training (p < 0.05).
FIGURE 5. Maximal mitochondrial enzyme activities pre and post high intensity
interval training. Values are mean ± SE, n = 8. -HAD, -hydroxy-acy-CoA
dehydrogenase; wm, wet mass. * Significantly higher pre training (p < 0.05).
FIGURE 6. FABPpm, FAT/CD36 and HSL protein content pre and post high intensity
Page 31 of 38

32
interval training. Values are mean ± SE, n = 8. FABPpm, plasma membrane fatty acid
binding protein; FAT/CD36, fatty acid translocase, HSL, hormone sensitive lipase. *
Significantly higher than pre training (p < 0.05).
Page 32 of 38

33
Figure 1
S5S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
HIIT
S5S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S5S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S5S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S5S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S5S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S4S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3S1
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3S1
VO2peak
test
60 min
@ 60%
VO2peak
S2S1
VO2peak
test
60 min
@ 60%
VO2peak
S2S1
VO2peak
test
60 min
@ 60%
VO2peak
VO2peak
test
VO2peak
test
60 min
@ 60%
VO2peak
S2 S3
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
60 min
@ 60%
VO2peak
VO2peak
test
S6 S7
Day 1 3 5 7 9 11 13 15 17 20 22-23
HIIT
Page 33 of 38

34
Figure 2
BOUT 1 BOUT 10
BOUT 3 BOUT 5
0
4
0
4
0
4
0
4
0
1
2
3
4Session 2
Session 7
*
*
Epinpehrine (nM)
Page 34 of 38

35
Figure 3
15 30 45 60
0
5
10
15
20
*
*
*
Pre-training
Post-training
Time (min)
Fat Oxidation (kJ
•
•
•
•
min
-1
)
Total ox CHO ox Fat ox
0
500
1000
1500
2000 Pre-training
Post-training
*
*
Total oxidation (kJ)
15 30 45 60
0
5
10
15
20
25
30
**
*
Time (min)
CHO Oxidation ( kJ
•
•
•
•
min
-1
)
Page 35 of 38

36
Figure 4
15 30 45 60
140
150
160
170
Pre-training
Post-training
*
*
Time (min)
Heart rate (beats
•
•
•
•
min-1)
0 15 30 60
0.0
0.3
0.6
0.9
1.2
1.5
*
*
Time (min)
Epinephrine (nM)
0 15 30 45 60
0.0
0.5
1.0
1.5
2.0
**
*
Time (min)
Plasma Lactate (mM)
Page 36 of 38

37
Figure 5

-HAD Citrate Synthase
0
5
10
15
20
25
30
35 PRE
POST
*
*
Maximal Enzyme Activity
(mmol




min
-1




kg wm
-1)
Page 37 of 38

38
Figure 6
FABP
pm FAT/CD36 HSL
0
50
100
150
*
PRE
POST
Total Protein Content
(Arbitrary units)
Page 38 of 38