J Physiol 595.

9 (2017) pp 2915–2930 2915

SYMPOSIUM REVIEW

Physiological adaptations to interval training and the role

of exercise intensity
Martin J. MacInnis and Martin J. Gibala
Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada

Training variables Training format Physiological adaptations

Frequency MICT

Skeletal muscle
Su M Tu W Th F Sa
X X X
X X X • Cellular stress
The Journal of Physiology

• Molecular responses
• Mitochondrial content
Intensity HIIT • Capillary density

Cardiovascular and integrative

Duration SIT • Maximum cardiac output

• Maximum stroke volume
• Blood volume
.
• VO2max

Abstract Interval exercise typically involves repeated bouts of relatively intense exercise inter-
spersed by short periods of recovery. A common classification scheme subdivides this method
into high-intensity interval training (HIIT; ‘near maximal’ efforts) and sprint interval training
(SIT; ‘supramaximal’ efforts). Both forms of interval training induce the classic physiological
adaptations characteristic of moderate-intensity continuous training (MICT) such as increased
aerobic capacity (V̇ O2 max ) and mitochondrial content. This brief review considers the role of
exercise intensity in mediating physiological adaptations to training, with a focus on the capacity
for aerobic energy metabolism. With respect to skeletal muscle adaptations, cellular stress and the
resultant metabolic signals for mitochondrial biogenesis depend largely on exercise intensity, with

Martin MacInnis is a Natural Sciences and Engineering Research Council of Canada (NSERC) Postdoctoral
Fellow in the Department of Kinesiology at McMaster University. His research is focused on exercise and
environmental physiology in humans, with a current emphasis on the molecular, metabolic and physiological
mechanisms regulating adaptations to exercise. His other research interests include altitude acclimatization,
nutrition, and exercise performance. Martin Gibala is Professor and Chair of the Department of Kinesiology
at McMaster University. He studies the regulation of skeletal muscle energy metabolism, including the impact
of exercise and nutrition on human health and performance. A prominent focus of his current research
programme is physiological adaptations to interval training in both healthy and diseased individuals. Both
authors are members of McMaster’s Exercise Metabolism Research Group.


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2916 M. J. MacInnis and M. J. Gibala J Physiol 595.9

limited work suggesting that increases in mitochondrial content are superior after HIIT compared
to MICT, at least when matched-work comparisons are made within the same individual. It is
well established that SIT increases mitochondrial content to a similar extent to MICT despite a
reduced exercise volume. At the whole-body level, V̇ O2 max is generally increased more by HIIT
than MICT for a given training volume, whereas SIT and MICT similarly improve V̇ O2 max despite
differences in training volume. There is less evidence available regarding the role of exercise
intensity in mediating changes in skeletal muscle capillary density, maximum stroke volume and
cardiac output, and blood volume. Furthermore, the interactions between intensity and duration
and frequency have not been thoroughly explored. While interval training is clearly a potent
stimulus for physiological remodelling in humans, the integrative response to this type of exercise
warrants further attention, especially in comparison to traditional endurance training.

(Received 28 July 2016; accepted after revision 11 October 2016; first published online 17 October 2016)
Corresponding author M. J. Gibala: Department of Kinesiology, McMaster University, 1280 Main St West, Hamilton,
ON, L8S 4K1, Canada. Email: [email protected]

Abstract figure legend Physiological responses to acute and chronic exercise are mediated by characteristics of the
training programme. Training volume is the product of the frequency, intensity, and duration of exercise. The format
of the training programme, although often complex, can generally be characterized as moderate-intensity continuous
training (MICT), high-intensity interval training (HIIT), or sprint interval training (SIT). Characteristics of the training
programme influence the magnitude of the skeletal muscle, cardiovascular and integrative adaptations to exercise.
In particular, there is strong evidence that exercise intensity mediates mitochondrial adaptations to exercise and
improvements in maximum aerobic capacity (V̇ O2 max ); however, the influence of exercise intensity is uncertain for
specific cardiovascular adaptations, including capillarization, maximum cardiac output and stroke volume, and blood
volume.

Abbreviations AMPK, AMP-activated protein kinase; CaMKII, Ca2+ /calmodulin-dependent protein kinase II; CS,
citrate synthase; CK, creatine kinase; COXIV, cytochrome c oxidase subunit IV; ETC, electron transport chain;
PHOS, glycogen phosphorylase; HIIT, high-intensity interval training; V̇ O2 max , maximum aerobic capacity; MICT,
moderate-intensity continuous training; OXPHOS, oxidative phosphorylation; V̇ O2 peak , peak aerobic capacity; p38
MAPK, p38 mitogen-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor γ 1-α; ROS, reactive
oxygen species; SIT, sprint interval training; SDH, succinate dehydrogenase; VEGF, vascular endothelial growth factor.

Introduction et al. 2012). This review will focus on the potential for
interval training to elicit physiological adaptations that
Exercise is traditionally defined as either endurance or enhance aerobic energy metabolism.
strength or viewed as a continuum anchored by these
common descriptors (Coffey & Hawley, 2007; Hawley Interval training terminology. Exercise prescription is
et al. 2014). In accordance with the principle of training obviously complex and involves numerous variables that
specificity, endurance training is associated with an can be manipulated, as evidenced by detailed reviews that
improved capacity for aerobic energy metabolism and have characterized interval training from the perspective
fatigue resistance, whereas strength training is linked of performance enhancement (Seiler, 2010; Tschakert
to muscle hypertrophy and increased force-generating & Hofmann, 2013; Buchheit & Laursen, 2013a,b). For
capacity (Baar, 2006; Egan & Zierath, 2013; Hawley et al. simplicity and consistency, this review will employ the
2014). Interval training, which can be simply defined as nomenclature put forward by Weston et al. (2014a) to
intermittent periods of intense exercise separated by peri- differentiate two basic types of interval training based on
ods of recovery (Fox et al. 1973), occupies a sort of middle exercise intensity (Fig. 1). High-intensity interval training
ground. Depending on the specific protocol employed, (HIIT) is defined as ‘near maximal’ efforts generally
this type of training can elicit adaptations resembling performed at an intensity that elicits  80% (but often
endurance or strength training, or a mix of the two. For 85–95%) of maximal heart rate. In contrast, sprint inter-
example, interval training using repeated Wingate tests val training (SIT) is characterized by efforts performed
is a potent stimulus to increase mitochondrial content at intensities equal to or greater than the pace that would
and peak aerobic capacity (V̇ O2 peak ; MacDougall et al. elicit V̇ O2 peak , including ‘all-out’ or ‘supramaximal’ efforts.
1998) while interval training using body-weight resistance The term moderate intensity continuous training (MICT)
exercise increases V̇ O2 peak and muscular strength (McRae is used for comparative purposes to describe exercise that is


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J Physiol 595.9 Training adaptations and the nature of the stimulus 2917

A Moderate-intensity B High-intensity
continuous training interval training
(MICT) (HIIT)
100 100
Total Total
duration duration
Workload (%PPO)

Workload (%PPO)
75 75

Avg. Avg.
50 50
Intensity Intensity

25 25

0 0
Time Time
C Sprint
interval training
(SIT)

100
Workload (%PPO)

75

Total Total
50 duration duration

25 Avg. Avg.
Intensity Intensity
0
Time Time (continued)

Low-volume
High-volume

D MICT HIIT
Low-volume High-volume
SIT SIT

Frequency: 3x/week 3x/week 3x/week 3x/week

Duration: 30 min 30 min 30 min 75 min
Mean intensity: 200 W 200 W 90 W 80 W

Weekly
training 1080 kJ 1080 kJ 486 kJ 1080 kJ
volume:

Figure 1. A graphical depiction of the main types of aerobic exercise

A–C, representative examples of moderate intensity continuous training (MICT), high-intensity interval training
(HIIT), and low and high volumes of sprint interval training (SIT). The intensity is depicted as a percentage of
the peak power output (PPO) obtained during a standard ramp V̇ O2 peak test (e.g. MacInnis et al. 2016). Note
that most recent studies of SIT used a low-volume protocol (e.g. Burgomaster et al. 2006, 2008), whereas earlier
studies of SIT used a high-volume protocol (e.g. Saltin et al. 1976). D, the training volume associated with each
protocol based on the durations and training frequencies provided. The MICT and HIIT protocols shown in A and
B are work-matched when performed for the same duration and at the same frequency. The low-volume SIT
protocol requires less total work to complete relative to HIIT and MICT, whereas performing three sessions of the
high-volume SIT protocol matches the training volume in the MICT and HIIT protocols.


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2918 M. J. MacInnis and M. J. Gibala J Physiol 595.9

performed in a continuous manner and at lower intensities A variety of methods are available for investigating
than HIIT. While imperfect, this general classification the effects of exercise on skeletal muscle mitochondria
scheme is nonetheless suitable for our purposes here. in humans. In general, acute studies measure changes
in the phosphorylation state of signalling proteins (e.g.
Assessing physiological adaptations to interval training. Ca2+ /calmodulin-dependent protein kinase II (CaMKII),
A fundamental and longstanding focus of exercise physio- AMP-activated protein kinase (AMPK), p38 mitogen
logy has been the elucidation of the mechanisms under- activated protein kinase (p38 MAPK)), gene expression
lying training adaptations. Of particular relevance to (e.g. peroxisome proliferator-activated receptor γ 1-α
the present discussion, improvements in aerobic energy (PGC-1α)), or mitochondrial protein synthesis rates.
metabolism are primarily linked to peripheral adaptations, Training studies usually assess the volume or area of
including increased skeletal muscle mitochondrial content mitochondria via microscopy, the activity or protein
and capillary density (Holloszy & Coyle, 1984), and central content of mitochondrial enzymes (e.g. citrate synthase
factors such as increased maximal stroke volume, maximal (CS) and succinate dehydrogenase (SDH)), or respiration
cardiac output and blood volume (Blomqvist & Saltin, in permeabilized muscle fibres or isolated mitochondria
1983; Bassett & Howley, 2000). These variables generally (e.g. oxidative phosphorylation (OXPHOS) capacity).
respond to changes in exercise volume, which is the While mitochondrial respiration is sometimes considered
product of exercise intensity (i.e. work per unit time), a measure of mitochondrial function and not content,
exercise duration (i.e. time per session), and training OXPHOS capacity is a biomarker of mitochondrial density
frequency (i.e. sessions per week), as shown in Fig. 1. (Larsen et al. 2012). Furthermore, enzyme activity (CS or
This review will consider the role of exercise intensity and COX) and OXPHOS capacity generally increase similarly
its interaction with duration and frequency in mediating in training studies, suggesting that mitochondrial function
physiological adaptations to interval training in humans. (i.e. respiration per unit of mitochondria) is not altered in
Given our primary expertise and the data available, the short-term (Jacobs et al. 2013; MacInnis et al. 2016). In
emphasis will be placed on skeletal muscle remodelling contrast, mitochondrial function correlates with aerobic
with a focus on the regulation of mitochondrial biogenesis. capacity in cross-sectional studies (Jacobs & Lundby,
A large body of research has evaluated the effect of 2013), indicating a potential long-term effect of training.
interval training on maximum aerobic capacity (V̇ O2 max ; The reader is directed elsewhere for detailed reviews on
Bacon et al. 2013; Gist et al. 2013; Sloth et al. 2013), the signalling pathways associated with mitochondrial
but data are limited regarding the underlying cardio- biogenesis (Scarpulla et al. 2012; Egan & Zierath, 2013)
vascular mechanisms involved. Finally, given our focus and the methodology available for assessing mitochondrial
on physiological adaptations in healthy (albeit sometimes responses to exercise in humans (Larsen et al. 2012; Miller
sedentary or overweight) populations, the reader is & Hamilton, 2012).
directed elsewhere for recent reviews regarding the effect The relatively rapid rate at which mitochondrial content
of interval training on health-related markers and disease responds to training permits relatively short-term studies
risk (Gibala et al. 2012; Kessler et al. 2012; Weston et al. of mitochondrial adaptations in humans. Similar to
2014a; Jelleyman et al. 2015; Ramos et al. 2015). MICT, a single session of HIIT or SIT activates signalling
pathways associated with mitochondrial biogenesis, such
Skeletal muscle adaptation to interval exercise as the phosphorylation of AMPK and p38 MAPK and
the expression of PGC-1α mRNA (Gibala et al. 2009;
training
Little et al. 2011; Metcalfe et al. 2015). The regular and
Mitochondrial responses to exercise and their time repeated activation of these pathways leads to increases
course. Skeletal muscle mitochondrial density regulates in mitochondrial density (Coffey & Hawley, 2007). A
substrate metabolism during submaximal exercise, with comprehensive study by Perry et al. (2010) examined the
increased mitochondrial content promoting a greater early time course of adaptations to HIIT and showed that
reliance on fat oxidation and a proportional decrease mRNA expression (e.g. PGC-1α) was acutely and trans-
in carbohydrate oxidation (Holloszy & Coyle, 1984; iently increased following each session of HIIT. CS protein
Egan & Zierath, 2013). As a result, exercise training content and enzyme activity increased steadily over the
lessens glycogen degradation and lactate production at a seven sessions, with CS maximal activity significantly
given intensity, while increasing the lactate threshold and increased above baseline after the third session. Other
allowing individuals to exercise for longer durations and studies have reported significant increases in CS activity
at greater percentages of their V̇ O2 max (Joyner & Coyle, 24 h after a single session of SIT (Little et al. 2011) or MICT
2008). Thus, given its central role in exercise performance, (Egan et al. 2013). Numerous studies have demonstrated
there is considerable interest in the factors mediating that mitochondrial content (measured with CS or COX
exercise-induced mitochondrial adaptations (Bishop et al. activity) increased by 25–35% after six to seven sessions
2014). of HIIT (Talanian et al. 2006; MacInnis et al. 2016) or


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J Physiol 595.9 Training adaptations and the nature of the stimulus 2919

SIT (Burgomaster et al. 2006; Gibala et al. 2006). When isolated mitochondria, which the authors hypothesized
the intensity and duration of exercise are held constant, was compensated through an increase in mitochondrial
mitochondrial content has been shown to plateau after content in the vastus lateralis muscle. Whether different
5 days of training (Egan et al. 2013); however, when the durations or intensities of exercise are capable of activating
intensity is increased progressively, mitochondrial content these ROS-dependent mechanisms to similar extents, or
continues to rise for at least several weeks (Henriksson & at all, remains unknown.
Reitman, 1977). The on-and-off pattern characteristic of interval
training (i.e. rest–work cycles) could partially explain
Exercise intensity mediates acute mitochondria-related skeletal muscle responses to this type of exercise.
responses to exercise. The molecular and cellular events AMPK phosphorylation was greater when a session of
that underpin adaptations to exercise are fundamental moderate-intensity exercise was divided into 1 min inter-
aspects of exercise biology (Egan & Zierath, 2013; Hawley vals, interspersed with rest, compared to when it was
et al. 2014), and the responsiveness of these pathways to performed as a continuous 30 min session (Combes et al.
divergent exercise stimuli is essential for understanding 2015); however, whether or not these acute differences
the process through which humans adapt to exercise (Baar, in signalling patterns would translate to different chronic
2006; Coffey & Hawley, 2007; Baar, 2009). Cellular stress effects is unclear. Research from our group suggests that
occurs in proportion to exercise intensity (Egan & Zierath, the intermittent nature of interval training plays a role in
2013), and there is strong evidence that higher intensities the magnitude of the adaptations. We demonstrated that
of exercise elicit a greater metabolic signal than moderate CS maximal activity was unchanged by performing a single
intensities (Fig. 2). Firstly, ATP turnover is greater for 4 min all-out bout of cycling 3 days week–1 for 6 weeks,
higher intensities of exercise (Howlett et al. 1998), which despite being increased in response to approximately the
also rely more on carbohydrate oxidation and utilize more same volume of work performed as four 30 s all-out
glycogen than do lower intensities of exercise (Gollnick bouts, interspersed with recovery periods (Cochran et al.
et al. 1974; Vøllestad & Blom, 1985; Romijn et al. 1993; 2014).
van Loon et al. 2001). Consequently, the accumulation
of intracellular lactate, creatine, AMP and ADP increases The role of exercise intensity in mediating mitochondrial
with exercise intensity (Howlett et al. 1998; van Loon et al. adaptations to training. Training volume has been
2001), as do the activity of AMPK (Wojtaszewski et al. suggested to be a primary determinant of the exercise-
2000; Egan et al. 2010; Kristensen et al. 2015) and CaMKII induced increase in mitochondrial content in humans
(Rose & Hargreaves, 2003; Rose et al. 2006; Egan et al. (Bishop et al. 2014). Supporting evidence in this regard
2010). The greater activation of these particular kinases, was derived mainly from correlations based on studies
which was elicited by high- compared to low-intensity that measured CS maximal activity before and after
exercise matched for total work, was associated with exercise programmes of different lengths rather than
greater expression of mRNA for PGC-1α, a major regulator work-matched HIIT and MICT protocols or studies
of mitochondrial biogenesis (Egan et al. 2010). Finally, and that compared relatively low volumes of SIT and high
downstream of the myriad metabolic signals described volumes of MICT. Given the limitations inherent to
above, mitochondrial protein synthesis was greater in making inferences based on a small pool of studies
response to continuous exercise performed at a higher with methodological differences, the authors called
intensity relative to work-matched exercise performed at a for additional research in humans comparing different
lower intensity (Di Donato et al. 2014), signifying a greater training stimuli within the same study. The training studies
rate of mitochondrial biogenesis when a given volume of included in this section are described in greater detail in
exercise is performed at a higher intensity. Table 1.
With respect to low-volume SIT in particular, two We recently examined the role of exercise intensity
recent studies suggest that the activation of mitochondrial in determining mitochondrial adaptations to short-term
biogenesis in response to all-out exercise may be training. Cognizant of the classic study design by Saltin
linked in part to the production of reactive oxygen and colleagues (1976), we employed single-leg cycling as
species (ROS; Fig. 2). Six bouts of Wingate-based SIT a model to examine the effect of two different training
induced ROS-dependent fragmentation of the ryanodine interventions within the same individual. Participants
receptor (RyR), which was implicated in the post-exercise performed six training sessions with each leg over
increase in intracellular Ca2+ concentration, a signal for 2 weeks, with one leg performing HIIT and the other
mitochondrial biogenesis (Place et al. 2015). Additionally, leg performing MICT (MacInnis et al. 2016). A weight
Larsen et al. (2016) reported that 2 weeks of Wingate-based was affixed to the contralateral crank arm during exercise,
SIT inhibited aconitase activity through an increase in providing the ‘feel’ of two-legged cycling, even though
ROS. The inhibition of this tricarboxylic acid cycle participants trained with only one leg at a time (Abbiss
enzyme was associated with reduced respiration in et al. 2011; Burns et al. 2014). Importantly, the volume


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2920 M. J. MacInnis and M. J. Gibala J Physiol 595.9

Sarcoplasm B ATP turnover

C Glycogenolysis, pyruvate
production/oxidation and
lactate accumulation

Glucose Lactate

PHOS HK LDH
ATP Cr ATP AMP
ATPase CK CK MK PFK
G-1-P G-6-P Pyruvate
ADP PCr ADP ADP

A Calcium release
D Accumulation
of free radicals, ions, PDH
Sarcoplasmic
reticulum and metabolites Acetyl-CoA
RyR Ca2+
TCA
Ca2+ Ca2+ La– ADP cycle
ROS OH AMP H2O2 [Citrate]
Ca2+
ROS ACN
Ca 2+
O2
– H +
Pi Cr
Ca2+ Ca2+
J Aconitase ?
inhibition
I RyR-mediated K Mitophagy
calcium leak
E Signalling
cascade activation
P P ?
Nucleus
AMPK CaMKII
Mitochondrion
F Mitochondrial gene
expression
H Mitochondrial content
PGC-1α
TF TF

NUGEMPs
ETC ETC

TF TF TFAM ETC

PPARGC1A
G Mitochondrial protein synthesis

Figure 2. A schematic diagram of the putative mechanisms through which high-intensity exercise may
elicit greater mitochondrial adaptations to aerobic training compared to lower intensities of exercise
Exercising at a higher intensity increases calcium release (A), requires greater ATP turnover (B), and leads to
greater use of carbohydrates for fuel (C), compared to exercising at a lower intensity. As a result, there is
a greater accumulation of metabolites, ions, and free radicals (D), which increase the activation of signalling
proteins (E), including the kinases Ca2+ /calmodulin-dependent protein kinase II (CaMKII) and AMP-activated
protein kinase (AMPK). The increased activity of these protein kinases causes greater rates of gene expression
for PGC-1α (encoded by PPARGC1A), which in turn acts as a transcriptional co-activator for nuclear genes
encoding mitochondrial proteins (NUGEMPs; F). In turn, mitochondrial protein synthesis rates are greater for
high-intensity exercise (G), leading to a greater increase in mitochondrial content (H), relative to exercise at a
lower intensity. Two additional ROS-mediated mechanisms explaining the potency of low-volume SIT have recently
been reported. Firstly, through a ROS-dependent mechanism, low-volume SIT led to the fragmentation of the
ryanodine receptor (RyR) of the sarcoplasmic reticulum and increased the intracellular calcium concentration (I),
a signal for mitochondrial biogenesis. Similarly, two weeks of low-volume SIT was associated with the inhibition
of aconitase in the tricarboxylic acid cycle (TCA) and an increased intracellular citrate concentration, which was
suggested to increase mitochondrial content via a reduction in mitophagy (J). For specific references, see ‘Exercise
intensity mediates acute mitochondria-related responses to exercise’ in text. ACN, aconitase; ATPase, adenosine
triphosphatase; CK, creatine kinase; ETC, electron transport chain; PHOS, glycogen phosphorylase; HK, hexokinase;
LDH, lactate dehydrogenase; MK, myosin kinase; PFK, phosphofructokinase; PDH, pyruvate dehydrogenase; TFAM,
transcription factor A, mitochondria.


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J Physiol 595.9

Table 1. Description of studies that have compared mitochondrial adaptations to different training protocols in healthy humans

Activity level; rel.

V̇ O2 max (ml kg−1 Mitochondria-related
Author n (M/F) min−1 ) Duration; frequency; mode Protocols measurements

Bækkerud et al. (2016) 12/18 NR; 34 6 weeks; 3 days week−1 ; running MICT: 45 min at 70% HRmax † CS activity
and walking HIIT: 10 min at 70% HRmax ; 4×
(4 min at 85–95% and 3 min at
70% HRmax )†

C 2016 The Authors. The Journal of Physiology 

SIT: 10× (1 min at 90% and
active recovery)†

Burgomaster et al. (2008) 10/10 Moderately active; 41 6 weeks; 3–5 days week−1 ; cycling MICT: 40–60 min at 65% CS activity, substrate oxidation
V̇ O2 peak
SIT: 4–6× (30 s Wingate test and
4.5 min at 30 W)

Daussin et al. (2008) 7/4 Sedentary; 30 8 week; 3 days week−1 ; cycling MICT: 20–35 min at 61% Wmax † Mass-specific respiration

C 2016 The Physiological Society

SIT: 4–7× (4 min at 54% and
1 min at 90% of Wmax )†

Gibala et al. (2006) 16/0 Moderately active; 50 2 week; 3 days week−1 ; cycling MICT: 90–120 min at 65% COX activity, ETC complex 4
V̇ O2 peak protein content
SIT: 4–6× (30 s Wingate test and
4.0 min at 30 W)

Gillen et al. (2016) 25/0 Sedentary; 32 12 week; 3 days week−1 ; cycling MICT: 2 min at 50 W; 45 min at CS activity, ETC complexes 1–5
Training adaptations and the nature of the stimulus

70% HRmax ; 3 min at 50 W protein content

SIT: 2 min at 50 W; 3× (20 s
‘modified’ Wingate test and
2 min at 50 W); 1 min at 50 W

Gorostiaga et al. (1991) 3/9 Moderately active or 8 week; 3 days week−1 ; cycling MICT: 30 min at 50% Wmax † CS activity
sedentary; 36 SIT: 30× (30 s at 100% Wmax and
30 s rest)†

(Continued)
2921
 2922

Table 1. Continued

Activity level; rel.

V̇ O2 max (ml kg−1 Mitochondria-related
Author n (M/F) min−1 ) Duration; frequency; mode Protocols measurements

Granata et al. (2015) 29/0 Moderately active; 46 4 week; 3 days week−1 ; cycling MICT: 20–36 min 65% Wmax † CS activity, ETC complexes 1–5
HIIT: 4–7× (4 min at 90% protein content, mass- and
Wmax and 2 min at 60 W)† mitochondria-specific
SIT: 4–10× (30 s Wingate test respiration
and 4 min rest)

Henriksson & Reitman 9/0 NR; 47 7-8 week; 3 days week−1 ; cycling MICT: 27 min at 79% V̇ O2 max † SDH activity
(1976) HIIT: 5× (4 min at 101% V̇ O2 max
and 2 min rest)†

MacInnis et al. (2016) 10/0 Moderately active; 46 2 week; 3 days week−1 ; cycling MICT: 5 min at 25 W; 30 min at CS activity, ETC complex 4
50% Wmax † protein content, mass- and
HIIT: 5 min at 25 W; 4× (5 min at mitochondria-
65% and 2.5 min at 20% specific respiration
Wmax )†

Saltin et al. (1976) 13/0 Not regularly training; 4 week; 4–5 days week−1 ; cycling MICT: 35–45 min at 75% SDH activity


46 single-leg V̇ O2 max †
SIT: 20–30× (40–50 s at 150%
single-leg V̇ O2 max and 90 s
rest)†
M. J. MacInnis and M. J. Gibala

Scribbans et al. (2014) 16/3 Moderately active; 48 6 week; 4 days week−1 ; cycling MICT: 30 min at 65% V̇ O2 max SDH activity
SIT: 8× (20 s at 170% V̇ O2 max
and 10 s rest)

Shepherd et al. (2012) 16/0 Sedentary; 42 6 week; 3–5 days week−1 ; cycling MICT: 40–60 min at 65% ETC complex 4 protein content,
V̇ O2 max substrate oxidation
SIT: 4–6× (30 s Wingate test and
4.5 min at 30 W)

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Note that the classic Wingate test consists of a 30 s ‘all-out’ cycling effort against a resistance equivalent to 7.5% of body weight, whereas the ‘modified’ Wingate test involved
a 20 s effort against a resistance of 5.0% of body weight. †Protocols that had the same oxygen cost or were work-matched. CS, citrate synthase; ETC, electron transport chain;
HRmax , maximum heart rate; HIIT, high-intensity interval training; MICT, moderate-intensity continuous training; NR, not reported; SDH, succinate dehydrogenase; SIT, sprint
interval training; V̇ O2 max , maximum aerobic capacity; Wmax , peak power output.
BMI and age ranged from 22-30 kg/m2 and 21-45 years, respectively

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J Physiol 595.9
J Physiol 595.9 Training adaptations and the nature of the stimulus 2923

of training was identical for each leg. We showed that of moderate-intensity exercise. Comparable increases in
HIIT compared to MICT elicited a greater increase in mitochondrial content were reported for low-volume SIT
mitochondrial content, assessed by CS maximal activity and MICT after 2 (Gibala et al. 2006), 6 (Burgomaster
and OXPHOS capacity in permeabilized muscle fibres et al. 2008; Shepherd et al. 2012; Scribbans et al. 2014)
(Fig. 3). Mitochondrial function (i.e. OXPHOS capacity and 12 (Gillen et al. 2016) weeks of training. In the
normalized to CS maximal activity) was unchanged with longest of these comparisons, we demonstrated that
training. three weekly sessions of SIT (1 min of intense exercise
Our recent findings are supported by a crossover study performed over a 10 min session) elicited similar increases
that compared work-matched HIIT and MICT in the in CS maximal activity compared to a MICT protocol
same individuals (Daussin et al. 2008). In that study, that involved 150 min of weekly exercise (Gillen et al.
the high-intensity programme elicited an increase in 2016). Furthermore, low-volume SIT induced similar
skeletal muscle mitochondrial respiration whereas the improvements relative to MICT for multiple aspects of
moderate-intensity programme did not. In the three fat and carbohydrate metabolism (Gibala et al. 2006;
comparisons of work-matched HIIT and MICT that Burgomaster et al. 2008; Shepherd et al. 2012; Scribbans
used parallel-group designs, increases in mitochondrial et al. 2014). When low-volume SIT was compared to
content were similar (Henriksson & Reitman, 1976; a higher volume of HIIT, an increase in mitochondrial
Bækkerud et al. 2016) or not observed (Granata et al. respiration was only apparent following SIT (Granata
2015). Work-matched comparisons addressing the effect et al. 2015). Interestingly, CS activity did not increase for
of intensity on lactate threshold generally report similar either condition in that study. Finally, low-volume SIT
increases across groups as well (Poole & Gaesser, 1985; increased lactate threshold, a variable strongly associated
Helgerud et al. 2007; Granata et al. 2015). Sample sizes with skeletal muscle mitochondrial content (Ivy et al.
were relatively small in all of the above work-matched 1980), to a similar extent relative to greater volumes of
studies; however, within-subject comparisons provide MICT and HIIT (McKay et al. 2009; Granata et al. 2015).
greater control for potential sources of variation (e.g. Observations from studies comparing high-volume SIT
diet, sleep, stress) and greater statistical power than and MICT suggest that there are diminishing returns
between-subject comparisons. We suggest that the lack of with increased durations of SIT (i.e. number of bouts per
consensus among these studies is partly due to differences session). Comparisons of work-matched SIT and MICT
in experimental design. resulted in similar increases in SDH maximal activity after
Supporting evidence for the role of exercise intensity 4 weeks (Saltin et al. 1976) or greater increases in CS
is derived from studies reporting that a small volume of maximal activity for MICT relative to SIT after 8 weeks
exercise performed at a very high intensity can elicit similar of training (Gorostiaga et al. 1991). The total duration
skeletal muscle adaptations compared to a large volume of sprint exercise performed in both of these studies

A MICT B
15 HIIT §† 100
(mmol • kg protein–1 • hr–1)

(pmol O2 • [s • mg ww]–1
Mass-specific PCl&Cll JO2

80 †
Maximal CS activity

10
60

40
5
20

0 0
PRE POST PRE POST
Training Training

Figure 3. Changes in mitochondrial content in response to 2 weeks of high-intensity interval training

(HIIT) and moderate-intensity continuous training (MICT), matched for total work
Subjects performed six sessions of single-leg cycling with each leg, completing either a HIIT or MICT protocol. The
greater increase in mitochondrial content elicited by HIIT as compared to MICT was evident from post-training
differences in maximal citrate synthase activity (A) and mitochondrial respiration (J O2 ), specifically oxidative
phosphorylation capacity through complexes I and II (PCI&CII , B). Bars represent the mean responses for each
group, whereas lines refer to the responses of individual subjects. Symbols indicate a significant difference from
the within-group, pre-training mean (§), and significant differences between groups at the post-training mean (†).
Error bars represent 1 standard error of the mean. For A, n = 9 and for B, n = 8 subjects.


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2924 M. J. MacInnis and M. J. Gibala J Physiol 595.9

(10–25 min; 20–30 bouts) was much higher than recent intensities. Similarly, performing SIT twice per week versus
studies (1–3 min; 3–6 bouts). Note that in both studies, once per week augmented the improvement in lactate
the two protocols elicited similar decreases in blood threshold following 6 weeks of training (Dalleck et al.
lactate concentrations during submaximal exercise, and 2010). The increase in CS maximal activity was also similar
in the latter study, CS maximal activity was numerically (Parra et al. 2000) or blunted (Hatle et al. 2014) when
greater in the SIT group post-training. The hypothesis exercise was performed at a high (7–8 sessions per week)
of diminishing returns with increased number of bouts compared to moderate frequency (2–3 sessions per week);
is supported by acute studies of SIT. Parolin et al. however, subjects performed the same number of sessions
(1999) demonstrated that AMP and ADP concentrations over different lengths of time across protocols, preventing
were greatest after the first of three Wingate tests, and conclusions related to the effect of training frequency.
glycogenolysis and lactate accumulation were strongly Recently, Granata et al. (2016) reported that
depressed during the third bout relative to the first bout increasing the volume of HIIT (by augmenting duration
of exercise, suggesting that the metabolic signal was not and frequency while maintaining intensity) increased
enhanced with further bouts. Furthermore, the expression mitochondrial content, providing evidence that increases
of genes related to mitochondrial biogenesis was similar in the volume of high-intensity exercise can augment
for SIT and MICT, whether work was matched (Wang et al. mitochondrial content; however, the relative importance
2009) or not (Psilander et al. 2010). of frequency or duration in mediating this adaptation
cannot be determined from this study.
The potential effects of exercise duration and frequency
on mitochondrial adaptation. Studies performed in The role of skeletal muscle recruitment pattern and
rodents have demonstrated that increasing the training fibre type. Evidence from rodent studies suggests that
volume by raising the duration (Fitts et al. 1975; Dudley mitochondrial adaptations to exercise occur in a fibre
et al. 1982) or frequency (Hickson, 1981) of exercise type-specific manner (Dudley et al. 1982; Taylor et al.
augments mitochondrial adaptations to aerobic exercise; 2005); however, the interaction between fibre type and
however, insufficient data are available to fully ascertain exercise intensity has received less attention in humans,
the roles of these variables in mediating mitochondrial as most studies examine adaptations at the whole-muscle
adaptations to exercise in humans. level. Skeletal muscle recruitment occurs in proportion
The effect of duration seems to depend on the intensity to exercise intensity (Vøllestad & Blom, 1985; Sale,
of the exercise. Green and colleagues (2012, 2013) 1987), implying that higher intensities of exercise could
examined ‘metabolic strain’ (a proxy for mitochondrial elicit greater responses in type II fibres relative to lower
content) during steady-state exercise following 10 days of intensities of exercise.
cycling for 30 or 60 min day–1 at a low, moderate, or In our recent comparison of work-matched HIIT and
high intensity (60, 70 and 86% of V̇ O2 max , respectively). MICT (MacInnis et al. 2016), we hypothesized that
Following training at the two higher intensities, but changes in mitochondrial content (measured with cyto-
not the lowest intensity, the accumulation of AMP and chrome c oxidase subunit IV (COXIV) protein content)
ADP and the depletion of phosphocreatine and glycogen would be greater in type II fibres following HIIT as
during steady-state exercise were reduced more following compared to MICT. We demonstrated an effect of training
the 60 min day–1 as compared to the 30 min day–1 on COXIV in whole muscle, but we were unable to
programmes, suggesting increases in mitochondrial demonstrate that response in either fibre type. In contrast,
content (Holloszy & Coyle, 1984). Thus, the effect of greater increases in SDH maximal activity in type II
duration was augmented at higher exercise intensities. fibres have been reported following HIIT relative to
In a relatively large comparison of two 11-week training work-matched MICT (Henriksson & Reitman, 1976),
programmes consisting of mixed exercise modes and and type II fibre activation and AMPK activity were
intensities, the high dose (3800 kcal week–1 ) and the greater following an acute session of HIIT relative to a
moderate dose (2000 kcal week–1 ) groups exhibited comparable session of MICT (Kristensen et al. 2015).
similar increases in markers of mitochondrial content In rodent muscle, increases in exercise intensity led to
(Reichkendler et al. 2013; Rosenkilde et al. 2015), which a plateau in mitochondrial content in red quadriceps
could be explained by the relatively low average intensity muscle, whereas relatively high intensities of exercise were
(67% of V̇ O2 max ). Note that neither study compared two necessary to increase the mitochondrial content of white
durations of interval training. quadriceps muscle (Dudley et al. 1982; Taylor et al. 2005).
Limited data are available to ascertain whether weekly Comparisons of low-volume SIT and MICT demonstrated
training frequency influences mitochondrial adaptations similar increases in COX expression (Shepherd et al. 2012)
in humans. Costill et al. (1991) reported a greater increase and SDH activity (Scribbans et al. 2014) across fibre types
in CS maximal activity for well-trained swimmers who despite the differences in training volume and the expected
trained twice each day versus once each day at high differences in muscle recruitment between SIT and MICT.


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J Physiol 595.9 Training adaptations and the nature of the stimulus 2925

Thus, data regarding the potential for different training as phlebotomizing subjects returned cardiac output and
programmes to induce fibre type-specific mitochondrial V̇ O2 max to baseline values (Bonne et al. 2014; Montero
responses to exercise in humans is inconclusive. et al. 2015a). Although plasma and blood volumes
increase after relatively few exercise sessions (Convertino
Interval training and skeletal muscle capillary density. et al. 1980; Green et al. 1987; Graham et al. 2016),
Skeletal muscle capillarization requires weeks to months contributing to increased stroke volume and decreased
to manifest in response to exercise training (Andersen heart rate during submaximal exercise (Green et al. 1990;
& Henriksson, 1977; Hoppeler et al. 1985), and changes Goodman et al. 2005), changes in maximum stroke
in capillary density appear to be blunted at higher volume and cardiac output seem to require more time to
exercise intensities (see Gliemann, 2016). Low-volume manifest. Improvements in maximum stroke volume have
SIT induced similar or greater increases in the expression been reported after 2–6 weeks of training in some (e.g.
of several angiogenesis-related mRNAs relative to MICT, Warburton et al. 2004; Esfandiari et al. 2013; Bonne et al.
including greater vascular endothelial growth factor 2014; Montero et al. 2015a) but not all training protocols
(VEGF) expression; however, the concentration of muscle (e.g. Macpherson et al. 2011; Jacobs et al. 2013).
interstitial VEGF protein and the proliferation of cultured
endothelial cells were lower following the SIT session The role of exercise intensity in mediating improvements
(Hoier et al. 2012). These acute differences corresponded in V̇ O2 max . In a meta-analysis comparing the effects of
with the increased and unchanged capillary density interval and continuous training on V̇ O2 max in healthy
following the 4-week preconditioning period of MICT and adults, Milanovic et al. (2016) reported a greater response
the 4 weeks of SIT, respectively. In the one comparison of to interval training relative to continuous training whether
work-matched HIIT and MICT we are aware of, skeletal training volume was equal or not. This conclusion is
muscle capillarization increases were greatest following supported by an analysis from Bell & Wenger (1988),
MICT (Daussin et al. 2008); however, two separate studies which demonstrated a linear improvement in V̇ O2 max as
reported similar increases in capillary density with 6 weeks training intensity increased from 50 to 100% of V̇ O2 max ,
of low-volume SIT or MICT (Cocks et al. 2013; Scribbans and a meta-analysis from Bacon et al. (2013), which
et al. 2014). With the limited data, it is difficult to reported a greater increase in V̇ O2 max for high-intensity
reconcile the inconsistent results, but in all cases MICT training relative to values typically reported in large studies
was more or equally as effective for increasing capillary of MICT. Similarly, a meta-analysis by Weston et al.
density compared to HIIT/SIT. To our knowledge, there (2014a) concluded that HIIT was more effective than
are no human data addressing the role of exercise duration work-matched MICT for improving V̇ O2 max in patients
or training frequency on skeletal muscle capillarization; with lifestyle-induced cardiometabolic disease. Finally,
however, given the relationship between capillary density a recent, large randomized control trial of different
and exercise performance (Coyle et al. 1988; Iaia et al. intensities of continuous exercise with obese adults
2011), this area of research is deserving of more attention. supports these meta-analyses: greater increases in V̇ O2 max
were demonstrated in response to 24 weeks of exercise
Cardiovascular adaptations to interval exercise performed at 75% of V̇ O2 max relative to isocaloric exercise
performed at 50% of V̇ O2 max , with differences apparent
training
after 8 weeks of training (Ross et al. 2015).
Time course of cardiovascular adaptations to exercise The importance of exercise intensity in improving
training in humans. Improvements in V̇ O2 max typically V̇ O2 max is further evident from comparisons of MICT and
manifest as early as 2–4 weeks after initiating training much lower volumes of SIT/HIIT. Low-volume SIT/HIIT
(Henriksson & Reitman, 1976; Andersen & Henriksson, performed for 2–16 weeks increased V̇ O2 max (Gist et al.
1977), but V̇ O2 max can increase after 1 week (Hickson 2013; Sloth et al. 2013; Weston et al. 2014b), with
et al. 1977). The latter study reported the largest mean improvements elicited by the high-intensity protocols
increase in V̇ O2 max in humans, a 44% increase over being equal to improvements from MICT protocols when
10 weeks in response to a high volume of intense inter- compared (Gist et al. 2013). In agreement with these
val and continuous training. In cross-sectional studies, analyses, we recently demonstrated that low-volume SIT
the variation in V̇ O2 max is predominately attributable to increased V̇ O2 peak to the same extent as MICT over a
variation in maximum stroke volume (and cardiac output) 12-week period, despite a fivefold difference in training
as opposed to the arteriovenous O2 difference (Bassett volume (Gillen et al. 2016).
& Howley, 2000; Montero et al. 2015b), and training Relatively few studies have investigated changes in
studies generally reach the same conclusion (e.g. Ekblom V̇ O2 max in response to different durations or frequencies of
et al. 1968). The increase in maximum cardiac output interval training; however, both variables appear to have
observed after several weeks of endurance training was relatively small effects on V̇ O2 max compared to the effect
related to exercise-induced haematological adaptations, of exercise intensity. For example, the improvements in


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2926 M. J. MacInnis and M. J. Gibala J Physiol 595.9

V̇ O2 max were not different in subjects who performed either or similar when the volume of interval training is
one or four bouts of 4 min intervals (Tjønna et al. 2013) lower. For other physiological variables, the effect of
or either 2 or 4 days per week of interval training (Fox intensity is unclear, and it is uncertain whether inter-
et al. 1975). In a series of classic studies for which subjects val training is advantageous compared to MICT. Given
trained for 10 weeks and then continued to perform a lower the relative lack of data regarding the influences of
volume of exercise for 15 weeks, Hickson and colleagues exercise duration and training frequency on physiological
reported that maintaining intensity (Hickson et al. 1985) adaptations to exercise, particularly for interval exercise,
was more important to preserve the training-induced more research is needed to understand how these training
increase in V̇ O2 max than maintaining duration (Hickson variables impact peripheral and central adaptations to
et al. 1982) or frequency (Hickson & Rosenkoetter, 1981). interval exercise. Specifically, we are unable to determine
While the effect of interval training duration on V̇ O2 max whether performing longer durations (i.e. a greater
is unclear, two meta-analyses suggested that longer inter- numbers of bouts per session) or greater frequencies
val bouts increased V̇ O2 max to a greater extent than of interval training would have beneficial effects for
shorter interval bouts (Bacon et al. 2013; Milanović et al. any of the variables in question. In summary, interval
2016). In contrast, Knuttgen et al. (1973) and Helgerud training is a powerful stimulus to elicit improvements
et al. (2007) both reported similar increases in V̇ O2 max in mitochondrial content and V̇ O2 max ; however, we
when comparable volumes of high-intensity exercise were know relatively little regarding the influences of exercise
performed as short (15 s) or long (3–4 min) intervals. intensity, duration, and frequency on other components of
the integrative physiological response to interval training.
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Shepherd SO, Cocks M, Tipton KD, Ranasinghe AM, Barker The authors have no competing interest to declare.
TA, Burniston JG, Wagenmakers AJM & Shaw CS (2012).
Sprint interval and traditional endurance training increase
net intramuscular triglyceride breakdown and expression of Author contributions
perilipin 2 and 5. J Physiol 591, 657–675.
Sloth M, Sloth D, Overgaard K & Dalgas U (2013). Effects of M.M. and M.G. conceived of the review, identified and inter-
sprint interval training on VO2max and aerobic exercise preted relevant studies for inclusion, wrote the manuscript,
performance: A systematic review and meta-analysis. Scand J and critically revised the manuscript. Both authors approved
Med Sci Sports 23, e341–e352. of the final manuscript and agreed to be accountable for all
Talanian JL, Galloway SDR, Heigenhauser GJF, Bonen A & aspects of the work. Both persons designated as authors qualify
Spriet LL (2006). Two weeks of high-intensity aerobic for authorship, and all those who qualify for authorship are
interval training increases the capacity for fat oxidation listed.
during exercise in women. J Appl Physiol 102, 1439–1447.
Taylor EB, Lamb JD, Hurst RW, Chesser DG, Ellingson WJ,
Greenwood LJ, Porter BB, Herway ST & Winder WW Funding
(2005). Endurance training increases skeletal muscle LKB1
and PGC-1α protein abundance: effects of time and Our laboratory is funded by an operating grant from the Natural
intensity. Am J Physiol Endocrinol Metab 289, E960–E968. Sciences and Engineering Research Council of Canada (NSERC;
Tjønna AE, Leinan IM, Bartnes AT, Jenssen BM, Gibala MJ, grant number RGPIN-2015-04632) to M.G., and M.M. is funded
Winett RA & Wisløff U (2013). Low- and high-volume of by an NSERC Postdoctoral Fellowship.
intensive endurance training significantly improves maximal
oxygen uptake after 10-weeks of training in healthy men.
PLoS One 8, e65382.


C 2016 The Authors. The Journal of Physiology 
C 2016 The Physiological Society