Three Minutes of All-Out Intermittent Exercise per Week Increases Skeletal Muscle Oxidative Capacity and Improves Cardiometabolic Health

Abstract

We investigated whether a training protocol that involved 3 min of intense intermittent exercise per week — within a total training time commitment of 30 min including warm up and cool down — could increase skeletal muscle oxidative capacity and markers of health status. Overweight/obese but otherwise healthy men and women (n = 7 each; age = 29±9 y; BMI = 29.8±2.7 kg/m2) performed 18 training sessions over 6 wk on a cycle ergometer. Each session began with a 2 min warm-up at 50 W, followed by 3×20 s “all-out” sprints against 5.0% body mass (mean power output: ∼450–500 W) interspersed with 2 min of recovery at 50 W, followed by a 3 min cool-down at 50 W. Peak oxygen uptake increased by 12% after training (32.6±4.5 vs. 29.1±4.2 ml/kg/min) and resting mean arterial pressure decreased by 7% (78±10 vs. 83±10 mmHg), with no difference between groups (both p<0.01, main effects for time). Skeletal muscle biopsy samples obtained before and 72 h after training revealed increased maximal activity of citrate synthase and protein content of cytochrome oxidase 4 (p<0.01, main effect), while the maximal activity of β-hydroxy acyl CoA dehydrogenase increased in men only (p<0.05). Continuous glucose monitoring measured under standard dietary conditions before and 48–72 h following training revealed lower 24 h average blood glucose concentration in men following training (5.4±0.6 vs. 5.9±0.5 mmol/L, p<0.05), but not women (5.5±0.4 vs. 5.5±0.6 mmol/L). This was associated with a greater increase in GLUT4 protein content in men compared to women (138% vs. 23%, p<0.05). Short-term interval training using a 10 min protocol that involved only 1 min of hard exercise, 3x/wk, stimulated physiological changes linked to improved health in overweight adults. Despite the small sample size, potential sex-specific adaptations were apparent that warrant further investigation.

Full text

Three Minutes of All-Out Intermittent Exercise per Week
Increases Skeletal Muscle Oxidative Capacity and
Improves Cardiometabolic Health
Jenna B. Gillen
1
, Michael E. Percival
1
, Lauren E. Skelly
1
, Brian J. Martin
1
, Rachel B. Tan
1
,
Mark A. Tarnopolsky
1,2
, Martin J. Gibala
1
*
1Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada, 2Department of Pediatrics and Medicine, McMaster University, Hamilton, Ontario, Canada
Abstract
We investigated whether a training protocol that involved 3 min of intense intermittent exercise per week — within a total
training time commitment of 30 min including warm up and cool down — could increase skeletal muscle oxidative capacity
and markers of health status. Overweight/obese but otherwise healthy men and women (n = 7 each; age = 2969 y; BMI
= 29.862.7 kg/m
2
) performed 18 training sessions over 6 wk on a cycle ergometer. Each session began with a 2 min warm-
up at 50 W, followed by 3620 s ‘‘all-out’’ sprints against 5.0% body mass (mean power output: ,450–500 W) interspersed
with 2 min of recovery at 50 W, followed by a 3 min cool-down at 50 W. Peak oxygen uptake increased by 12% after
training (32.664.5 vs. 29.164.2 ml/kg/min) and resting mean arterial pressure decreased by 7% (78610 vs. 83610 mmHg),
with no difference between groups (both p,0.01, main effects for time). Skeletal muscle biopsy samples obtained before
and 72 h after training revealed increased maximal activity of citrate synthase and protein content of cytochrome oxidase 4
(p,0.01, main effect), while the maximal activity of b-hydroxy acyl CoA dehydrogenase increased in men only (p,0.05).
Continuous glucose monitoring measured under standard dietary conditions before and 48–72 h following training
revealed lower 24 h average blood glucose concentration in men following training (5.460.6 vs. 5.960.5 mmol/L, p,0.05),
but not women (5.560.4 vs. 5.560.6 mmol/L). This was associated with a greater increase in GLUT4 protein content in men
compared to women (138% vs. 23%, p,0.05). Short-term interval training using a 10 min protocol that involved only 1 min
of hard exercise, 3x/wk, stimulated physiological changes linked to improved health in overweight adults. Despite the small
sample size, potential sex-specific adaptations were apparent that warrant further investigation.
Citation: Gillen JB, Percival ME, Skelly LE, Martin BJ, Tan RB, et al. (2014) Three Minutes of All-Out Intermittent Exercise per Week Increases Skeletal Muscle
Oxidative Capacity and Improves Cardiometabolic Health. PLoS ONE 9(11): e111489. doi:10.1371/journal.pone.0111489
Editor: Naoyuki Hayashi, Tokyo Institute of Technology, Japan
Received April 25, 2014; Accepted September 5, 2014; Published November 3, 2014
Copyright: ß2014 Gillen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This project was supported by a Natural Sciences and Engineering Research Council (NSERC) operating grant and McMaster University Internally
Sponsored Research Grant to MJG. JBG held a NSERC Vanier Canada Graduate Scholarship. MEP was supported by a NSERC Canada Graduate Scholarship
(Masters). LES held a NSERC undergraduate student research award and NSERC Canada Graduate Scholarship (Masters). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: gibalam@mcmaster.ca
Introduction
Interval exercise is characterized by repeated bursts of relatively
intense effort, interspersed by periods of rest or lower-intensity
exercise for recovery. Short-term interval training protocols can
induce physiological remodeling similar to continuous moderate-
intensity training, despite reduced time commitment and a
relatively small total exercise volume [1]. Recent studies have
also shown improvements in various health indices including
markers of glycemic control in both healthy individuals [2–4] and
people with cardiometabolic disorders including type 2 diabetes
[5] after low-volume interval training. These studies have been
conducted on relatively small numbers of subjects and involved
relatively short training interventions. Nonetheless, the findings
have garnered significant interest from a public health perspective,
given one of the most commonly cited barriers to regular exercise
participation is ‘‘lack of time’’ [6].
A common interval training model is the Wingate Test, which
involves a 30 s ‘‘all out’’ burst of cycling on a specialized
ergometer. Typically, 4–6 such intervals are performed, separated
by ,4–5 min of recovery, with three training sessions performed
each week [1]. Despite the very small total amount of exercise, a
training session typically lasts ,25 min, given the brief warm-up
and cool down that are usually included in addition to the
recovery periods. The relative ‘‘time efficiency’’ of Wingate-based
training has therefore been questioned [7], considering the
,75 min time commitment per week, which falls within the
physical activity guidelines advocated by some public health
agencies. While 150 min of moderate-intensity exercise per week is
the general recommendation [8,9] some guidelines include 75 min
of vigorous physical activity as an alternative [9].
Several recent studies investigated physiological and health-
related adaptations to very low-volume interval training protocols
that involved a time commitment of #15 min per session [10–12].
For example, Metcalfe and colleagues [10] reported that a 10 min
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training protocol, involving low-intensity cycling except for 2,
20 sec all out sprints, improved cardiorespiratory fitness (VO
2
peak) in previously sedentary adults when performed 3x/wk for
6 wk. The potential for very low-volume interval training
protocols to improve VO
2
peak has also been described by Ma
et al. [11] and Hazell et al. [12]. Metcalfe et al. [10] also reported
that insulin sensitivity based on oral glucose tolerance tests was
improved after training in men but not women, highlighting the
potential for sex-based differences in the adaptive response. Only
one study has examined muscle adaptations to this type of
training, with Ma et al. [11] reporting increased protein content of
some mitochondrial enzymes after training, although the maximal
activity of citrate synthase was unchanged.
The purpose of the present study was to clarify and advance our
understanding of the impact of very low-volume interval training
on physiological and health related adaptations to very low-
volume SIT. Specifically, we examined the impact of a training
protocol that involved only 1 minute of intense intermittent
exercise within a 10 min time commitment, including warm-up
and cool-down. Sedentary but otherwise healthy subjects trained
3x/wk for 6 wk, and needle biopsies were obtained before and
after training to examine skeletal muscle remodeling. We also
assessed changes in several markers reflective of cardiometabolic
health. In light of the findings by Metcalfe et al. [11], a secondary
aim was to explore potential sex-based differences in the adaptive
response to this type of training. We hypothesized that the training
intervention would increase skeletal muscle oxidative capacity, as
reflected by the maximal activity and protein content of
mitochondrial enzymes, increase VO
2
peak, and reduce resting
blood pressure and 24 h mean blood glucose concentration
measured using continuous glucose monitoring (CGM) under
conditions of controlled activity and feeding. We further hypoth-
esized that reductions in 24 h glucose would be superior in men.
Materials and Methods
The protocol for this study and supporting TREND checklist
are available as supporting information; see Checklist S1 and
Protocol S1.
Subjects
Fourteen overweight or obese men and women were recruited
by poster advertisement from the McMaster University commu-
nity and took part in the study (Table 1). Subjects were deemed
sedentary based on their self-reported habitual physical activity,
which consisted of #2 sessions/wk of structured exercise lasting #
30 min. Participants were allocated into the male or female
intervention group and matched for age, body mass index and
VO
2
peak. The experimental protocol, which consisted of
familiarization and baseline testing, a 6 wk training intervention,
and post-training measurements, was approved by the Hamilton
Integrated Research Ethics Board and all visits took place at
McMaster University. All subjects provided written informed
consent prior to their participation.
Experimental Protocol
Familiarization and baseline testing. Participants report-
ed to the laboratory on four separate occasions over 14 d for
familiarization and baseline testing during May-July 2013. On the
first visit, subjects initially sat quietly for 10 min prior to 3 separate
measurements of blood pressure using an automatic blood
pressure cuff (Contec 08A, Qinhuangdao, China), with the lowest
of these values used for analysis as previously reported [13].
Subjects subsequently performed an incremental maximal oxygen
uptake (VO
2
peak) test on an electronically braked cycle ergometer
(Lode Excalibur Sport V 2.0, Groningen, The Netherlands) as
previously described [14,15]. Briefly, following a 2 min warm-up
at 50 W, the resistance was increased by 1 W every 2 s until
volitional exhaustion or the point at which pedal cadence fell
below 50 rpm. A metabolic cart with an on-line gas collection
system (Moxus modular oxygen uptake system, AEI Technologies,
Pittsburgh, PA) acquired oxygen consumption (VO
2
) and carbon
dioxide production (VCO
2
) data. VO
2
peak was defined as the
highest average oxygen consumption over a 30 s period.
Approximately 15 min following the VO
2
peak test, participants
performed 1–2620 s all-out sprints on an electronically braked
cycle ergometer (Veletron, RacerMate, Seattle, WA, USA) to
become acquainted with the interval protocol.
Approximately 5 d after the familiarization session, participants
returned to the laboratory and were fitted with a continuous
glucose monitor (CGM; CGMS; iPro, Medtronic, Northridge,
CA) and chest-worn accelerometer (Actiheart; Camntech, Cam-
bridge, United Kingdom). Subjects were also given a glucose
meter (OneTouch UltraMini, Lifescan, Milpitas, CA) with
instructions on how to perform capillary blood sampling.
Participants received a standardized food parcel, which they were
instructed to consume at prescribed meal times over the
subsequent 24 h. The diet was individualized for each participant
and energy intake was estimated using the Mifflin-St Jeor equation
[16]. Mean total energy was 26236123 and 18866146 kcal for
men and women, respectively, derived from 5661% carbohy-
drate, 3061% fat and 1461% protein.
Starting at 600 h the day following CGM insertion, participants
began consuming the control diet under free-living conditions and
CGM data was collected for a 24 h period. Participants obtained
capillary blood glucose samples at four points over the 24 h period
when blood glucose was expected to be stable (i.e. upon
awakening, before lunch, before dinner and before bed) and were
automatically stored in the glucose meter provided. Average blood
glucose concentration, glucose area under the curve (AUC) and
the daily peak glucose concentration (G
max
) were calculated from
CGM data for a 24 h period from 600 to 559 h before and after
training. Physical activity was monitored continuously over this
24 h period using a chest-worn device (Actiheart) that simulta-
neously measured heart rate and activity with an internal
accelerometer that senses the frequency and intensity of torso
movements to calculate energy expenditure. Following CGM
removal at ,1200 h, glucose data were uploaded as previously
described [5].
Approximately 2 d later, participants reported to the lab
following a 10 h overnight fast. A single resting blood sample
was obtained by venipuncture from an antecubital vein. Plasma
and serum were separated by centrifugation (10 min at 4000 rpm)
and stored at 220uC for subsequent analysis. A resting skeletal
muscle biopsy was obtained using procedures we have previously
described [17]. Briefly, muscle samples were obtained from the
vastus lateralis under local anesthesia (1% lidocaine) using a
Bergstrom needle adapted with suction. Samples were sectioned
into several pieces, immediately snap frozen in liquid nitrogen and
stored at 280uC for later analysis.
Training protocol. At least 5 d following the muscle biopsy,
subjects initiated the interval training program, which consisted of
18 supervised sessions over 6 wk during June-August 2013.
Training was performed on Monday, Wednesday and Friday
each week. Each session consisted of 3620 s all-out cycling efforts
against a load corresponding to 0.05 kg/kg body mass, separated
by 2 min of low intensity cycling (50 W), on an electronically
braked ergometer (Veletron, RacerMate, Seattle, WA, USA). All
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training sessions included a 2 min warm-up and 3 min cool-down
at 50 W, for a total time commitment of 10 min. The weekly
training protocol therefore involved a total of 3 min of very intense
intermittent exercise within a time commitment of 30 min
including warm-up, cool-down and the recovery between efforts.
Peak and mean power output was recorded for each sprint and an
average determined for each session. Heart rate (HR) was
measured continuously on the first training session.
Post-testing. Resting blood pressure was measured 24 h after
the final training session, before subjects were fitted with the CGM
and Actiheart. CGM data was collected for a 24 h period starting
,48 h after the final exercise session and diet was controlled to be
the same as baseline. There was no difference in activity counts
between the baseline and post-testing CGM period (P.0.05).
Fasting blood and a resting muscle biopsy sample were obtained
72 h following the last training bout. Approximately 4 d following
the biopsy and 1 wk after the final training session, a maximal
exercise test was performed using the same procedures as at
baseline. All procedures and controls were identical to those
employed during baseline testing and took place during July-
September 2013. A flow chart of all participants involved in the
trial is depicted in Figure 1.
Blood Analysis
Plasma glucose was analyzed using a kit assay (Pointe Scientific,
Canton MI, USA) and serum insulin was measured by ELISA
according to manufacturer instructions (ALPCO Immunoassays,
Salem NH, USA). Insulin resistance was calculated using HOMA-
IR [18].
Muscle Analysis
Enzyme activity. One piece of muscle (,25 mg) was
homogenized in Lysing Matrix D tubes (MP Biomedicals, Solon,
OH, USA) using the FastPrep-24 Tissue and Cell Homogenizer
(MP Biomedicals, Solon, OH, USA) for 1065 s cycles at a speed
of 4 m/s with samples placed on ice for 5 min in between cycles.
Samples were homogenized in 20 volumes of buffer containing
70 mM sucrose, 220 mM mannitol, 10 mM HEPES supplement-
ed with protease inhibitors (Complete Mini, Roche Applied
Science, Laval, PQ, Canada) and used to determine the maximal
activity of citrate synthase and 3-b-hydroxyacyl CoA dehydroge-
nase (b-HAD) as we have previously described [14,19,20]. Protein
concentration of homogenates was determined using a commercial
assay (BCA Protein Assay, Pierce, Rockford, IL, USA) and
enzyme activity is expressed as mmol/kg protein/hr.
Western blotting. A second piece of muscle (,30 mg) was
homogenized in RIPA buffer using Lysing Matrix D tubes (MP
Biomedicals, Solon, OH, USA) with the FastPrep-24 Tissue and
Cell Homogenizer (MP Biomedicals, Solon, OH, USA). Samples
were processed for 4620 s cycles at 4.0 m/s, with samples placed
on ice for 5 min in between cycles, followed by 2620 s cycles at
4.0 m/s, with samples placed on ice for 2 min in between cycles.
Western blot analysis was conducted using techniques described
previously [14,19]. Briefly, protein concentration of homogenates
was determined (BCA Protein Assay) and equal amounts of
protein were prepared in 46Laemmli’s buffer and heated to 95uC
before being separated by 10% SDS-PAGE and electrotransferred
to nitrocellulose membranes. Ponceau S staining was performed
following transfer to visualize equal loading and transfer.
Following 1 h blocking in 5% fat-free milk Tris-buffered saline
0.1% Tween 20 (TBS-T), membranes were incubated in the
primary antibody (glucose transporter 4; Millipore, AB1345 or
COXIV; Mitosciences, MS408) overnight at 4uC in 3% fat-free
milk TBS-T based on previously optimized conditions. After
365 min washes in TBS-T, membranes were incubated in the
species-specific secondary antibody diluted (1:10,000) in 3% fat-
free milk TBS-T for 1 h at room temperature, washed in TBS-T
for 665 min, and visualized by chemiluminescence (SuperSignal
West Dura, Pierce) using a FluorChem SP Imaging System (Alpha
Innotech Corporation, San Leandro, CA, USA). ImageJ software
(NIH) was used to quantify the optical density of protein bands.
Protein content was expressed as a fold change relative to pre-
training for all subjects. a-tubulin (Cell Signaling Technology,
#2125), which did not change following training (p = 0.91), was
used as a loading control.
Statistical Analysis
All data were analyzed using a two-factor analysis of variance
(ANOVA), with the between factor group (men, women) and the
within factor time (pre-, post-training) using SPSS Statistics
software. Significant interactions and main effects were subse-
quently analyzed using a Tukey’s honestly significant difference
post hoc test. The level of significance for all analyses was set at
P,0.05 and all data are presented as means 6S.D for n = 7 in
each group, except for the CGM data which represents n = 6 per
group.
Results
Descriptive characteristics of training
Adherence to the training sessions was 100%. Mean HR,
measured continuously during the first training session and
Table 1. Subject Characteristics.
VARIABLE MEN WOMEN
Age (y) 296930610
Height (cm) 17665 16268
Weight (kg) 976875612*
Body Mass Index (kg/m
2
)31622962
VO2peak (L/min) 3.060.5 2.060.2*
VO2peak (ml/kg/min) 31642864
Maximal Workload (W) 262630 202623*
Values are means 6S.D. N = 7 for men and women. VO2peak, maximal oxygen uptake.
*Significantly different from men (p#0.05).
doi:10.1371/journal.pone.0111489.t001
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averaged over the entire 10 min protocol including warm-up and
cool-down, was 8362% of HR
max
. Relative PPO and MPO
measured on the first and last training session did not differ
between men and women and increased with training (Table 2,
main effect for time, p,0.01). The average HR response for all
subjects and average MPO for men and women during session 1 is
depicted in Figure 2.
Figure 1. Flow diagram of participants through all phases of the trial.
doi:10.1371/journal.pone.0111489.g001
Table 2. Markers of Health and Fitness.
MEN WOMEN
VARIABLE PRE POST PRE POST
Body Mass (kg) 77612 77613 79615 79615
FPG (mmol/L) 5.160.3 5.260.3 5.060.3 5.060.3
FPI (uIU/ml) 13.567.9 10.767.0* 9.664.0 7.163.0*
HOMA-IR 3.161.9 2.561.5* 2.160.9 1.560.6*
Gmax (mmol/L) 8.061.3 6.861.1* 7.360.6 7.660.9
Resting SBP (mmHg) 1246811668* 109611 100611*
Resting DBP (mmHg) 71611 676566696069
Resting MAP (mmHg) 88688364* 80610 7469*
Relative PPO (W/kg FFM) 11.364.1 12.263.6* 10.060.6 11.861.1*
Relative MPO (W/kg FFM) 9.061.6 10.661.5* 9.060.5 12.060.1*
Values are means 6S.D. N = 7 for men and women. *Significantly different than pre-training (p#0.05).
FPG, fasting plasma glucose; FPI, fasting plasma insulin; Gmax: daily peak glucose concentration.
doi:10.1371/journal.pone.0111489.t002
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Skeletal muscle adaptations to very low-volume SIT
The maximal activity of citrate synthase increased by ,40%
after training (Fig. 3A, main effect for time, p,0.001). COXIV
protein content also increased after training with no differences
between groups (Fig. 3B, main effect for time, p,0.01), however
b-HAD maximal activity only increased after training in the men
(Fig. 3C; interaction between training and sex, p,0.05). GLUT4
protein content increased in both men and women following
training (Fig. 4A, main effect for time, p,0.01), however men
increased to a greater extent compared to women (138% vs. 23%,
interaction between training and sex, p,0.05).
Indices of cardiometabolic health
Very low-volume interval training increased VO
2
peak by 12%
in both men and women (Fig. 5, main effect for time, p,0.001),
which was associated with a 14% increase in maximal workload
(Table 1, main effect for time, p,0.001). Mean arterial pressure
(MAP) was reduced by 6% and 8% in men and women
respectively following training (Table 2, main effect for time, p,
0.01). Systolic blood pressure (SBP) was also reduced following
training (Table 2, main effect for time, P,0.01), while diastolic
blood pressure (DBP) trended towards being lower (Table 2,
p = 0.07). Insulin sensitivity measured by HOMA-IR was
improved after training (Table 2, main effect for time, p,0.05),
owing mainly to a decrease in fasting serum insulin (Table 2, main
effect for time, p#0.05). There was no change in fasting plasma
glucose in either group (Table 2, p.0.05). CGM revealed a lower
24 h average blood glucose concentration after training in men
(5.460.6 vs. 5.960.5 mmol/L, p,0.05) but not women (Fig. 4B,
Figure 3. Very low-volume SIT improves skeletal muscle mitochondrial capacity. Measured in muscle biopsy samples obtained from the
vastus lateralis before (PRE) and 72 h after (POST) 6-week SIT in men and women. Maximal activity of citrate synthase (A), protein content of COXIV (B)
and maximal activity of b-HAD (C). Values are means 6SD (n = 7 per group). Representative Western blots for 2 men and 2 women are shown for
COXIV. a-tubulin was used a loading control and representative Western blots are shown. *P,0.05, pre- vs. post-training; +p,0.05, men vs. women at
same time point; line denotes a main effect.
doi:10.1371/journal.pone.0111489.g003
Figure 2. Characterization of the low-volume SIT protocol. Solid
line represents average heart rate (HR) response, expressed as a % of
maximum, for all subjects during the first training session (left side y-
axis). Bar graph represents relative mean power output (MPO) per
kilogram fat-free mass (FFM) for men (dark bar) and women (white bar)
during the first training session (right side y-axis).
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5.560.6 vs. 5.560.4 mmol/L, p.0.05). Similarly, 24 h glucose
AUC was reduced in men only (Fig. 4C, interaction between
training and sex, p,0.05). G
max
was lower in men following
training, but not in women (Table 2, interaction between training
and sex, p,0.01)
Discussion
The main finding from the present study was that short-term
interval training, using a protocol that involved only 1 min of very
intense exercise within a total time commitment of 10 min, was a
potent stimulus to induce physiological adaptations that are linked
to improved health in overweight and obese adults. Our general
design, which involved 3 sessions per week for 6 wk, was similar to
recent studies by Metcalfe [10] and Ma [11], but clarified
outstanding questions regarding the potential for very low-volume
interval training to increase muscle oxidative capacity, resting
blood pressure and aspects of glycemic control. Despite the small
sample size, we also found evidence of potential sex-specific
adaptations to this type of training that warrant further
investigation.
Very low-volume interval training increases muscle
oxidative capacity
A recent systematic review and meta-analysis [21] proposed a
classification scheme for interval training in an effort to
Figure 4. Improved indices of blood glucose control in men following very low-volume SIT. GLUT4 protein content measured in muscle
biopsy samples obtained from the vastus lateralis before (PRE) and 72 h after (POST) 6 week SIT in men and women (A). Individual 24 h average
blood glucose concentration (B) and 24 h blood glucose area under the curve (AUC) measured before (PRE) and 48–72 h after (POST) 6 week SIT in
men and women using continuous glucose monitoring (CGM). Values are means 6SD (n = 7 per group for muscle data, n =6 per group for CGM
data). Representative Western blots for 2 men and 2 women are shown for GLUT4. *P,0.05, pre- vs. post-training; +p,0.05, men vs. women at same
time point.
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standardize terminology employed in future studies. Using the
descriptors proposed by Weston and colleagues, we have opted to
classify the present protocol as ‘‘sprint interval training’’ (SIT)
given the ‘‘all-out’’ efforts, as opposed to ‘‘high-intensity interval
training’’ (HIIT), which the authors define as bouts performed at
relatively intense but nonetheless submaximal workloads corre-
sponding to 80–100% of maximal heart rate [21]. We report here
for the first time that very low-volume SIT can increase the
maximal activity of citrate synthase, which is reportedly one of the
best indicators of mitochondrial content in human skeletal muscle,
as it is highly correlated with gold-standard measures made by
electron microscopy [22]. While skeletal muscle adaptations to
SIT are well established, there are limited and equivocal data
regarding the effect of very low-volume SIT on mitochondrial
content. Skleryk et al. [23] reported that a protocol involving 8–
12610 s all-out cycle sprints against 5% body weight interspersed
with 80 s rest, performed six times over 2 wk, did not improve
mitochondrial capacity in overweight men as reflected by a lack of
change in the protein content of COXII and COXIV. In contrast,
Ma et al. [11] showed that a protocol consisting of 8, 20 s cycling
efforts at an intensity of 170% of VO
2
peak and interspersed with
10 s of recovery, performed four times per week for 4 wk,
increased the protein content of COXI and COXIV, however, the
maximal activity of citrate synthase was unchanged. The results
from the present study confirm that 6 wk of very low-volume SIT,
involving a total of only 3 min of all out intermittent exercise
within a 30 min time commitment per week, was a sufficient
stimulus to elicit a robust increase in citrate synthase similar to
what has been reported after protocols involving a larger volume
of SIT or traditional moderate-intensity continuous training that
involve a much greater total volume of exercise and training time
commitment [24]. Clearly, there is some minimal total volume of
SIT necessary to acutely stimulate mitochondrial biogenesis,
which when performed repeatedly leads to measureable increases
in enzyme protein content or maximal activity. The various short-
term, very low-volume SIT protocols that have been employed to
date are likely on the lower end of this threshold, which may in
part explain the equivocal results to date. Additional studies, like
the elegant work by Perry et al. [25], which characterized the early
time course of adaptation to HIIT, will help to resolve this matter.
Effect of very low-volume interval training on markers of
cardiometabolic health
Seminal work by Tabata and colleagues over two decades ago
showed that 7–8 bouts of 20 s all out sprints, with 10 s rest in
between, improved VO
2
peak in young men by 15%, when
performed four times per week for 6 wk [26]. The beneficial effect
of ‘‘Tabata style’’ training on VO
2
peak, which is a popular
exercise strategy among many personal trainers, was recently
confirmed by Ma et al. [11] who reported a 19% increase in young
men after 4 wk. The present work, and recent studies by others
[10–12,27], confirm that very brief bouts of all-out exercise,
performed a few times per week, is a very time efficient strategy to
improve VO
2
peak, which is a strong predictor of all-cause
morbidity and mortality [28]. A novel, important finding from the
present work was the significant reduction in MAP when
measured 24 h after the final training bout, which is of similar
magnitude to findings following traditional Wingate-based SIT in
overweight/obese men and women [13], as well as 16 wk of high
volume aerobic interval or continuous moderate intensity training
in individuals with metabolic syndrome [29]. Based on findings
from a recent systematic review and meta-analysis, the blood
pressure reduction in the present study is of similar magnitude to
those following intermittent isometric resistance training [30],
which is emerging as a very effective exercise strategy for lowering
resting blood pressure [30,31]. It is unclear if our findings
represent an acute effect of the last training bout, however if one
performs SIT every other day as in the present study, the
beneficial effect on blood pressure would be maintained.
Potential sex-specific adaptations to low-volume interval
training
SIT has been shown to improve insulin sensitivity, based on
hyperinsulinemic euglycemic clamps performed on sedentary and
recreationally active individuals [2] as well as oral glucose
tolerance tests (OGTTs) performed on young healthy [4] and
overweight/obese [13] men. Metcalfe et al. [10] recently reported
that a 10 min low-intensity cycling protocol that included 2620 s
all-out sprints, performed 18 times over 6 wk, improved insulin
sensitivity measured by OGTTs in men but not women [10].
Consistent with the observations of Metcalfe et al. [10], we found
using CGM that 24 h average blood glucose concentration,
glucose AUC and G
max
, measured under standard dietary
conditions from 48–72 h after the final training session, were
improved in men but not women. Interestingly, the training-
induced increase in total GLUT4 protein content was approxi-
mately 6-fold higher in men compared to women.
The lack of change in peripheral glucose control in women in
the present study is consistent with recent reports by others
[10,15], although this is not a universal finding [2]. It is possible
that our low sample size (n = 6 for CGM data), or the fact that the
women had higher 24 h blood glucose control at baseline,
influenced our findings and resulted in a type 2 statistical error.
Nonetheless, by way of a possible related mechanism, it has also
been speculated that the rapid improvement in insulin sensitivity
following SIT is attributed to high rates of glycogen breakdown
and subsequent re-synthesis following each exercise bout [10], and
women have been shown to break down 42% less muscle glycogen
in type I fibers during a single Wingate sprint [32]. Future studies
are needed to investigate if GLUT4 translocation following acute
SIT is blunted in women, and definitively determine in larger
cohorts of subjects if improvements in glucose control following
SIT are sex-specific. HOMA-IR was improved in both men and
women after training, owing to significant reductions in fasting
plasma insulin, and consistent with previous studies [3,33].
We also found sex-specific differences in a marker of lipid
oxidation capacity, based on changes in the maximal activity of b-
HAD which were detected in men but not women. A similar
period of Wingate-based SIT was reported to improve the
Figure 5. Very low-volume SIT increases VO
2
peak. Measured
before (PRE) and 1 week following (POST) 6 wk SIT in men and women.
Values are means 6SD (n = 7 per group). *P,0.05, pre- vs. post-
training; line denotes a main effect.
doi:10.1371/journal.pone.0111489.g005
Three Minutes of Intense Exercise per Week Improves Health
PLOS ONE | www.plosone.org 7 November 2014 | Volume 9 | Issue 11 | e111489

maximal activity of b-HAD in both men and women, but that
study did not involve a specific comparison between sexes [24].
Similar to the pre-training CGM data, it is possible that the higher
baseline value for b-HAD in women in the present study reduced
their potential to increase the capacity for lipid oxidation
compared to men. Other recent studies however, have also
highlighted sex-based differences in the skeletal muscle adaptive
response to SIT in active young men and women [34]. Scalzo et
al. [34] reported higher rates of muscle protein synthesis in men
compared to women following a 3 wk SIT intervention, based on
oral administration of deuterium oxide. Future research is needed,
using designs which control for menstrual cycle phase and initial
fitness level [35], to evaluate if sex-based differences exist in the
skeletal muscle adaptive response low-volume SIT.
Conclusions
In summary, we report that 3 min of all-out exercise performed
within a 30 min time commitment per week including warm-up
and cool-down, improved skeletal muscle oxidative capacity and
indices of cardiometabolic health including VO
2
peak and blood
pressure, in overweight/obese adults. The protocol employed in
the present study involved a training time commitment that was
considerably lower than in previous Wingate-based SIT studies
(i.e., 10 versus ,25 min per session) and provides further evidence
of the potential for very brief, intense bursts of exercise to elicit
physiological adaptations that are associated with improved health
status in a time-efficient manner. Despite the small sample size,
potential sex-specific adaptations were apparent that warrant
further investigation.
Supporting Information
Checklist S1 TREND checklist.
(PDF)
Protocol S1 Trial study protocol.
(DOCX)
Acknowledgments
We would like to thank Dr. Jonathan Little and Dr. Michael Riddell for
their assistance in helping to facilitate the continuous glucose monitor
measurements.
Author Contributions
Conceived and designed the experiments: JBG MJG. Performed the
experiments: JBG MEP LES BJM RBT MAT MJG. Analyzed the data:
JBG MEP LES BJM RBT. Contributed reagents/materials/analysis tools:
MAT MJG. Wrote the paper: JBG MJG.
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