ORIGINAL RESEARCH
Unchanged content of oxidative enzymes in fast-twitch
muscle ﬁbers and _VO2 kinetics after intensiﬁed training in
trained cyclists
Peter M. Christensen1,2, Thomas P. Gunnarsson1, Martin Thomassen1, Daryl P. Wilkerson3,
Jens Jung Nielsen1 & Jens Bangsbo1
1 Department of Nutrition, Exercise and Sports, Section of Integrated Physiology, University of Copenhagen, Copenhagen, Denmark
2 Team Danmark (Danish Elite Sport Organization), Copenhagen, Denmark
3 Sport and Health Sciences, St Luke’s Campus, University of Exeter, Exeter, UK
Keywords
_VO2 kinetics, high intensity training, interval
training, type I ﬁbers, type II ﬁbers.
Correspondence
Jens Bangsbo, Department of Nutrition,
Exercise and Sports, Section of Integrated
Physiology, University of Copenhagen,
August Krogh Building, Universitetsparken
13, 2100 KBH Ø, Denmark.
Tel: +45 35 32 16 23
Fax: +45 35 32 16 00
E-mail: jbangsbo@nexs.ku.dk
Funding Information
The study was supported by Team Danmark
(Danish Elite Sport Organization).
Received: 8 May 2015; Accepted: 19 May
2015
doi: 10.14814/phy2.12428
Physiol Rep, 3 (7), 2015, e12428,
doi: 10.14814/phy2.12428
Abstract
The present study examined if high intensity training (HIT) could increase
the expression of oxidative enzymes in fast-twitch muscle ﬁbers causing a fas-
ter oxygen uptake ( _VO2) response during intense (INT), but not moderate
(MOD), exercise and reduce the _VO2 slow component and muscle metabolic
perturbation during INT. Pulmonary _VO2 kinetics was determined in eight
trained male cyclists ( _VO2-max: 59  4 (means  SD) mL min1 kg1) dur-
ing MOD (205  12 W ~65% _VO2-max) and INT (286  17 W ~85% _VO2-
max) exercise before and after a 7-week HIT period (30-sec sprints and 4-min
intervals) with a 50% reduction in volume. Both before and after HIT the
content in fast-twitch ﬁbers of CS (P < 0.05) and COX-4 (P < 0.01) was
lower, whereas PFK was higher (P < 0.001) than in slow-twitch ﬁbers. Con-
tent of CS, COX-4, and PFK in homogenate and fast-twitch ﬁbers was
unchanged with HIT. Maximal activity (lmol g DW1 min1) of CS (56  8
post-HIT vs. 59  10 pre-HIT), HAD (27  6 vs. 29  3) and PFK
(340  69 vs. 318  105) and the capillary to ﬁber ratio (2.30  0.16 vs.
2.38  0.20) was unaltered following HIT. _VO2 kinetics was unchanged with
HIT and the speed of the primary response did not differ between MOD and
INT. Muscle creatine phosphate was lower (42  15 vs. 66  17 mmol kg
DW1) and muscle lactate was higher (40  18 vs. 14  5 mmol kg DW1)
at 6 min of INT (P < 0.05) after compared to before HIT. A period of inten-
siﬁed training with a volume reduction did not increase the content of oxida-
tive enzymes in fast-twitch ﬁbers, and did not change _VO2 kinetics.
Introduction
It has been known for decades that endurance training
results in a faster increase in the pulmonary oxygen
uptake ( _VO2) response in the initial phase of exercise
(Hickson et al. 1978). During constant load exercise at
intensities above the gas exchange threshold (GET) _VO2
continues to rise at a slow rate, with training reported to
reduce the magnitude of this ‘ _VO2 slow component’
(Jones et al. 2011). These alterations may be due to ele-
vated muscle oxidative enzyme capacity and greater oxy-
gen delivery (Jones and Poole 2005) as have been found
in training studies using untrained subjects with a maxi-
mal _VO2 ( _VO2-max) of ~50 mL min1 kg1 (Saltin et al.
1976; Phillips et al. 1995; Shoemaker et al. 1996; Krustrup
et al. 2004a). In the study of Krustrup et al. subjects per-
formed high intensity training (1 min intervals) and it
was reported that post-training leg _VO2 kinetics was fas-
ter during intense but not moderate exercise when com-
pared to pretraining, respectively (Krustrup et al. 2004a).
This may have been caused by adaptations in fast-twitch
(FT) muscle ﬁbers recruited during training, since these
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License,
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2015 | Vol. 3 | Iss. 7 | e12428
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Physiological Reports ISSN 2051-817X
ﬁbers have been reported to have lower oxidative maxi-
mal enzyme activity than slow-twitch (ST) muscle ﬁbers
(Essen et al. 1975; Essen-Gustavsson and Henriksson
1984; Schantz and Henriksson 1987). In support for
training causing ﬁber type-speciﬁc adaptations, maximal
activity of oxidative enzymes in FT muscle ﬁbers can
reach the same level as ST ﬁbers in highly trained athletes
( _VO2-max: ~70 mL min1 kg1) (Jansson and Kaijser
1977; Chi et al. 1983) and a training study with untrained
subjects observed an increase in maximal activity in pools
of FT ﬁbers following intense training (Henriksson and
Reitman 1976).
Training
studies
using
untrained
subjects
typically
result in several adaptations. Therefore, it cannot be
determined if one adaptation (e.g., increase in oxidative
enzyme capacity) is of importance for the faster rise in
_VO2 at the onset of exercise and the alterations in the
_VO2 slow component when other adaptations also occur
in response to training (e.g., increase in bulk oxygen
delivery or reduced mean transit time at the capillary level
due to increased capillary density). Instead, studying the
effect of an altered training regime of trained subjects
could potentially provide insight into what factors are
important to improve the _VO2 kinetics since fewer mus-
cular and vascular adaptations are expected to occur. Fur-
thermore, just a few studies have been conducted with
trained athletes and some (Dufour et al. 2006; Christen-
sen et al. 2011), but not all studies (Norris and Petersen
1998; Demarle et al. 2001), reported that _VO2 kinetics
remained unaltered. However, comparison is difﬁcult due
to various designs used ranging from high-volume train-
ing for 8 weeks at moderate intensity at the start of the
season in cyclists (Norris and Petersen 1998), 6 weeks in
runners encompassing both moderate and intense bouts
(~90% _VO2-max in hypoxia or normoxia) (Dufour et al.
2006), to intense training (<5 min) either with a reduc-
tion in volume in soccer players for 2 weeks at the end of
the season (Christensen et al. 2011) or a maintained
training volume in runners for 8 weeks (Demarle et al.
2001).
Recruitment of FT ﬁbers has been implicated in the
development of the _VO2 slow component (Barstow et al.
1996; Jones et al. 2011). Thus, it may be that an increased
content of oxidative enzymes in FT ﬁbers could reduce
the _VO2 slow component in trained athletes since a high
proportion of FT ﬁbers has been associated with a large
slow component (Barstow et al. 1996) and since FT ﬁbers
appear to have a lower maximal oxidative enzyme activity
than ST ﬁbers (Essen et al. 1975; Essen-Gustavsson and
Henriksson 1984; Schantz and Henriksson 1987) unless
vigorous training has been performed (Jansson and Kaij-
ser 1977; Chi et al. 1983). Moreover, since oxidative
enzymes are thought to be implicated in the rate of the
rise in _VO2 at the onset of exercise (Jones and Poole
2005), it may be that intense training targeting both ST
and FT ﬁbers is essential for eliciting faster _VO2 kinetics
in trained athletes. This argument is supported by an aug-
mented signaling for cascades involved in the synthesis of
oxidative enzymes in well-trained cyclists ( _VO2-max:
68 mL min1 kg1) following repeated 30-sec sprinting
compared with less intense exercise (Psilander et al.
2010). Also, in less trained subjects repeated 30-sec
sprinting activated signal cascades involved in the synthe-
sis of oxidative enzymes to a similar extent as long dura-
tion low intensity exercise (Little et al. 2010, 2011). The
response of these signaling cascades increases in an inten-
sity-dependent manner (Egan et al. 2010; Nordsborg et al.
2010), which could be related to recruitment of more
muscle ﬁbers. In runners subjected to intense training in
combination with a marked lowering of low- and moder-
ate
intensity
training
(~25–50%)
maximal
oxidative
enzyme activity in muscle homogenate was maintained
(Bangsbo et al. 2009; Iaia et al. 2009) which could be due
to adaptations in FT ﬁbers since detraining is known to
lower activity of oxidative enzymes (Chi et al. 1983;
Christensen et al. 2011). Thus, repeated 30-sec sprinting
separated by ~4 min of rest may be an effective training
regime to cause increases in oxidative enzymes in FT
ﬁbers. The majority of studies in untrained subjects
(Jacobs et al. 1987; MacDougall et al. 1998; Burgomaster
et al. 2005, 2008; Gibala et al. 2006) did not evaluate ﬁber
type-speciﬁc adaptations in oxidative enzymes, but recent
studies do suggest that repeated 20–30 sec sprints can
increase markers of oxidative capacity in both ﬁber types
in untrained subjects (Shepherd et al. 2013; Scribbans
et al. 2014). However, such evaluation has not been per-
formed on trained individuals following intense training
with 30-sec sprints (Bangsbo et al. 2009; Iaia et al. 2009;
Christensen et al. 2011; Gunnarsson et al. 2012). Oxida-
tive capacity is higher in ST than FT ﬁbers (Essen et al.
1975; Essen-Gustavsson and Henriksson 1984; Schantz
and Henriksson 1987, Shepherd), which means that the
potential for improvements likely is greater in the FT
ﬁbers supported by similar capacity in the two ﬁber types
in highly trained subjects (Jansson and Kaijser 1977; Chi
et al. 1983).
A faster _VO2 response is associated with reductions in
the anaerobic contribution to the total energy turnover at
the onset of exercise. This has been determined from
muscle metabolites and blood lactate either as a result of
performing prior exercise (Bangsbo et al. 2001; Krustrup
et al. 2001) or performing exercise training either with
low intensity and high volume (Phillips et al. 1995; Green
et al. 2009) or high intensity and low volume (Burgomas-
ter et al. 2006). However, limited knowledge exists about
muscle anaerobic energy turnover after a period of high
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ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Metabolic and Muscle Adaptations to HIT
P. M. Christensen et al.
intensity training of trained subjects. Venous lactate when
running at intensities below
_VO2-max was unchanged
after 4 weeks of repeated 30-sec sprint training in moder-
ately trained runners ( _VO2-max: 55 mL min1 kg1)
(Iaia et al. 2009) and after 6–9 weeks of combined aero-
bic high intensity training and repeated 30-sec sprinting
in
trained
runners
( _VO2-max:
61 mL
min1 kg1)
(Bangsbo et al. 2009), with both studies encompassing a
reduction
in
weekly
training
distance.
Changes
in
blood lactate likely reﬂect changes at the muscular level
(Green et al. 2009) but neither of the studies evaluated
changes in muscle lactate and creatine phosphate during
exercise. Following 3 weeks with high-intensity training
(8 9 5 min) added to the normal training, cyclists ( _VO2-
max: 65 mL min1 kg1) had lower muscle lactate but
unchanged
creatine
phosphate
content
directly
after
intense exercise (Clark et al. 2004). Therefore, it is
presently unknown if high intensity training encompass-
ing
repeated 30-sec
sprint
intervals,
in
combination
with reduction in volume, changes muscle anaerobic
energy turnover during submaximal exercise in trained
subjects.
Thus, the aims of the present study, using a training
intervention consisting of repeated 30-sec sprinting and
aerobic high intensity training for a group of trained
cyclists, were to examine (1) whether intensiﬁed training
increases the amount of oxidative enzymes in FT muscle
ﬁbers as well as speeds _VO2 kinetics and reduces the _VO2
slow component during intense exercise; (2) if the anaer-
obic energy turnover during intense exercise was reduced
by training. We hypothesized that the intensiﬁed training
would increase the oxidative capacity of FT ﬁbers result-
ing in faster _VO2 kinetics during intense exercise, but not
during moderate intensity exercise, as well as reduce the
_VO2 slow component and anaerobic energy turnover dur-
ing intense exercise.
Methods
Subjects
Eight trained male cyclists with an average (SD) age,
weight, and _VO2-max of 33  8 years, 81  8 kg, and
4.8  0.3 L min1 or 59  4 mL min1 kg1, respec-
tively, were recruited for the study. Prior to participating
in the study, the subjects had trained/competed ~three to
ﬁve times each week for at least 3 years. The study proce-
dures were approved by the local ethical committee of the
capital region of Copenhagen (Region Hovedstaden) and
all subjects received written and oral information about
the study procedures and gave their written informed
consent to participate in the study in accordance with the
Helsinki declaration.
Experimental design
The subjects carried out a 7-week high-intensity training
(HIT) intervention (see later) from October to December
just after the season had ﬁnished, hence subjects were
expected to be ﬁt and in a physical stable condition. Both
before and after HIT the subjects carried out two main
experiments (EXP1 & EXP2) to evaluate changes in the met-
abolic response and _VO2 kinetics during submaximal exer-
cise (< _VO2-max). The subjects and training intervention
were the same as in a study focusing on adaptations of ion
transport proteins, ion kinetics, and performance during
repeated high intensity exercise (Gunnarsson et al. 2013).
Training
Subjects performed four supervised training sessions per
week on their own bikes on public roads. Training was
performed as 12 9 30-sec uphill (~6% gradient) maximal
sprints interspersed with 4–5 min low intensity recovery
(SPR; 2.59 week1) resulting in a 1:8–10 work rest ratio,
and 5 9 ~4 min aerobic high intensity intervals separated
by ~2 min of rest (AEH; 1.59 week1) on a ﬂat 2.5 km
course with a work rest ratio of 2:1. In a training week
day 1 was recovery, AEH was performed on day 2, SPR on
day 3, recovery on day 4, SPR on day 5, recovery on day
6, and ﬁnally SPR or AEH on day 7 in alternate weeks. To
ensure maximal effort during training drafting was not
allowed and both SPR (1 vs. 1) and AEH (mass start) was
performed in a competitive manner with the objective of
ﬁnishing ﬁrst. Heart rate (HR) was measured in 5-sec
intervals during training (Polar Team Edition, Finland).
Peak-HR during each SPR interval was 90  4% of HR-
max and average-HR during each AEH interval was
89  2% of HR-max. The physiological response from
one of the subjects during a SPR and an AEH training ses-
sion is shown in Fig. 1. Weekly volume was ~240 min
during HIT (15 min SET [~6%], 30 min AEH [~12%],
135 min low intensity recovery between intervals [56%]
and ~60 min moderate intensity [25%] as transport to
and from training). This amounted to a ~50% reduction
of the training volume being 472  153 min week1
before HIT (0–60% HR-max [29%], 60–70% HR-max
[24%], 70–80% HR-max [19%], 80–90% HR-max [21%],
90–95% HR-max [6%], 95–100% HR-max [1%]).
Exercise testing
All testing was performed on a mechanically braked
ergometer bike (Monark 839E, Varberg, Sweden) with the
subjects using their own pedals and speciﬁc geometric
setup which was maintained throughout the study. Sub-
jects were instructed not to perform any training the day
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 7 | e12428
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P. M. Christensen et al.
Metabolic and Muscle Adaptations to HIT
before a testing session, and maintain the same food
intake and abstain from intake of caffeine on days of test-
ing.
During the ﬁrst visit to the laboratory during the com-
petitive season subjects performed an incremental test
starting out at 100 W with increments of 25 W min1
until exhaustion. Pulmonary _VO2 was measured breath by
breath (Oxycon Pro, Viasys Healthcare, CareFusion, Rolle,
Switzerland) and VO2-max and HR-max was determined
as the highest value over a 30-sec period. In addition incre-
mental test peak power output was calculated as:
iPPO ¼ Power output (W) at the last completed stage
þ sec at the stage leading to task failure 60 sec1
 25 W:
iPPO was 409  24 W. Before and after HIT changes in
pulmonary _VO2 kinetics was investigated using repeated
exercise transitions. Subjects performed both moderate
(MOD: 50% iPPO, 205  12 W) and intense (INT: 70%
iPPO, 286  17 W) exercise with 2 min at 20 W pre-
ceding all intervals. The absolute exercise intensity was
maintained throughout the study. Both prior to and fol-
lowing HIT subjects performed four transitions with
MOD each lasting 6 min (EXP1, EXP2, and two addi-
tional transitions on separate days) and three transitions
with INT each lasting 6 min (EXP1, EXP2, and an addi-
tional transition on a separate testing day) as well as two
transitions with INT lasting 3 min (EXP1, EXP2). Pul-
monary _VO2 was measured breath by breath. To deter-
mine _VO2 kinetics, errant breaths, deﬁned as any value
lying more than 4 SDs away from the local mean (e.g.,
due to swallowing and coughing) were initially removed.
Then the _VO2 responses in each intensity domain were
linearly interpolated to give 1-sec values, and then aver-
aged. The initial cardiodynamic component was ignored
by eliminating the ﬁrst 20 sec of data after the onset of
exercise.
MOD was modeled via a mono exponential function:
_VO2ðtÞ ¼ Baseline þ APð1  eðtTdP=sPÞÞ
INT was modeled via a bi-exponential function:
_VO2ðtÞ ¼Baseline þ APð1  eðtTdP=sPÞÞ
þ ASð1  eðtTdS=sSÞÞ
with _VO2 (t) being _VO2 to a given time (sec). _VO2 base-
line was calculated as average _VO2 from 30 to 90 sec of
0
4
8
12
16
20
1
2
3
4
5
0
4
8
12
16
20
1
2
3
4
5
6
7
8
9
10
11
12
Venous lactate
(mmol/L)
500
650
800
950
Power 
(W)
300
325
350
375
400
0
1000
2000
3000
4000
5000
6000
6
12
18
24
30
Power 
(W)
Time (min)
INTERVAL #
(12 x 30 sec; 5 min recovery)
INTERVAL #
(5 x 4 min; 2 min recovery)
VO2
(mL/min)
Venous lactate
(mmol/L)
A
B
C
D
Figure 1. The physiological response during a training session with 12 9 30-sec sprint intervals separated by 5 min of recovery (A & B) and a
session with 5 9 4 min intervals separated by 2 min of recovery (C & D) for one subject having a _VO2-max of 5.2 L min1 (hatched line). Peak
power (dotted bars), mean power (gray bars), and pulmonary _VO2 (full line) is shown for each interval (top) together with lactate (bottom)
from an antecubital vein before (open bars) and after (full bars) intervals. Each of the two training sessions was performed indoor using the
bike ergometer and _VO2-system described in the methods section.
2015 | Vol. 3 | Iss. 7 | e12428
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ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Metabolic and Muscle Adaptations to HIT
P. M. Christensen et al.
the 120 sec baseline cycling at 20 W. AP, TdP, and sP are
the amplitude, time delay, and time constant, respectively,
for the primary (P) response. AS, TdS, and sS are the
truncated amplitude, time delay, and time constant,
respectively, for the slow component (S). An iterative
process was used to determine the best ﬁt of the curve.
The relative VO2 slow component was determined as the
ratio between AS and average VO2 during the last 2 min
of exercise.
Mechanical gross efﬁciency (GE) was calculated for
both MOD and INT during the last 2 min of exercise
using the formula
GE ¼ external bike load ðkJ min1Þ
energy turnover ðkJ min1Þ  100%
Energy
turnover
was
estimated
as
_VO2
(L
min1) 9 energetic value of oxygen (kJ L1) with the lat-
ter
being
calculated
from
the
measured
respiratory
exchange ratio (RER) thus taking into account the differ-
ent energy yield from oxidation of carbohydrate and fat.
In the event of RER exceeding 1.0 a value of 1.0 was used
in the calculation.
Main experiments
Two experimental days (EXP1 & EXP2) were performed
both prior to and after HIT to quantify muscle metabo-
lites during INT. Subjects arrived to the laboratory in the
morning after consuming a light breakfast.
On both EXP1 and EXP2 a biopsy at rest was collected
from m. vastus lateralis under local anesthesia using a
Bergstrom needle with suction. One part was frozen
immediately in liquid nitrogen within ~10 sec for analysis
of metabolites, enzyme activity, and protein content.
Another part of the biopsy was embedded in tissue tec
(Sakura Finetek, Netherlands) for histochemical analysis.
Initially, 6 min of MOD was performed followed by rest
for 30 min. To evaluate changes in muscle metabolism in
response to the HIT-period a muscle biopsy was obtained
following 6 min of INT on EXP1 and following 3 min of
INT on EXP2. Following 60 min of rest, 3 min of INT
was performed on EXP1 and following 30 min of rest,
6 min of INT was performed on EXP2.
Muscle analysis
All muscle samples were stored at 80°C until analyzed.
Muscle metabolites
The muscle biopsies taken at rest and after 3 and 6 min
of INT were analyzed for levels of lactate and creatine
phosphate (CP) using ﬂuorometric methods (Lowry and
Passonneau 1972).
Maximal enzyme activity
In part of the muscle biopsy obtained at rest maximal
enzyme activity of citrate synthase (CS), 3-Hydroxyacyl
CoA dehydrogenase (HAD), phosphofructokinase (PFK),
and lactate dehydrogenase (LDH) was quantiﬁed in mus-
cle homogenates after freeze drying and removal of fat
and connective tissue using ﬂuorometric methods (Flu-
oroscan Ascent, Thermo Scientiﬁc, Waltham, MA) (Lowry
and Passonneau 1972).
Protein expression in muscle homogenate lysates
Approximately 3 mg freeze dried muscle tissue was split
in two for double protein determination to increase mea-
suring sensitivity and then homogenized and centrifuged
to
exclude
non
dissolved
structures,
as
previously
described (Bangsbo et al. 2009). Total protein concentra-
tions were determined in each sample using BSA stan-
dards (Pierce, IL) and the lysates were then diluted in 69
Laemmli buffer and ddH2O to reach equal protein con-
centration before protein expression of CS, cytochrome c
oxidase complex 4 (COX-4) and PFK were determined by
western blotting. For subsequent analysis the average
value was calculated from the two samples.
Protein expression in segments of human single
muscle ﬁbers
The determination of ﬁber type-speciﬁc changes in pro-
tein expression for CS, COX-4, and PFK were performed
as previously described Thomassen et al. (2013) with
minor changes. After freeze drying the muscle tissue sam-
ples (7–10 mg dry weight, n = 16) for 48 h, segments of
single ﬁbers were dissected under a microscope and
stored in single microfuge tubes. The average size of the
segments collected were roughly determined by measuring
the
lengths
of
the
ﬁber
under
a
microscope
(1.4  0.3 mm, mean  SD, n = 398). Before SDS-PAGE
18 lL 69 Laemmli buffer (0.7 mL 0.5 mol L1 Tris-base,
3 mL glycerol, 0.93 g DTT, 1 g SDS and 1.2 mg brom-
ophenol blue) diluted (1:3, v:v) in ddH2O was added to
each ﬁber and incubated for 1 h at room temperature.
In order to have equal number of ﬁbers in the different
groups, all single ﬁber segments were ﬁrst ﬁber typed
before the analysis of the protein of interest. About 5 lL
of the samples were loaded on a 26 well Tris-Tricine 4–
15% Criterion gels (Bio-Rad Laboratories, Solna, Sweden)
and by western blotting characterized as either slow
twitch (ST) or fast twitch (FT) muscle ﬁbers by use of
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 7 | e12428
Page 5
P. M. Christensen et al.
Metabolic and Muscle Adaptations to HIT
antibodies speciﬁc for myosin heavy chain (MHC) type I
(ST ﬁbers) and MHC type II (FT ﬁbers) as well as the
FT-speciﬁc SERCA1 protein (Thomassen et al. 2013).
Antibodies used were ST ﬁbers: 0.5 lg∙mL1, mouse
monoclonal IgM, A4.840, Developmental Studies Hybrid-
oma Bank (DSHB), University of Iowa, USA; FT ﬁbers:
2 lg∙mL1, mouse monoclonal IgG, A4.74, DSHB, both
developed by Dr Blau, and SERCA1: 0.1 lg∙mL1, mouse
monoclonal, MA3-912, Thermo Scientiﬁc. From this preﬁ-
ber typing, 160 ﬁbers were selected (1.5  0.3 mm) to the
ﬁnal analyses, including 20 from each subject with ﬁve
ﬁbers from each of the four groups: ST pre, ST post, FT
pre, and FT post. The remaining 13 lL of the given selected
ﬁber segments were then loaded onto additional gels.
Western blotting
For both segments of single muscle ﬁbers and muscle
homogenate lysates proteins were separated by SDS-PAGE
(55 mA per gel and maximum 150 V) for ~120 min and
then semi-dry transferred to a PVDF membrane (Milli-
pore A/S, Copenhagen, Denmark) for 120 min at 70 mA
per gel and maximum 25 V. After protein transfer, gels
including single ﬁbers were incubated for 1 h in Coomas-
sie stain, including 0.3% Coomassie Brilliant Blue R
(Sigma-Aldrich, Copenhagen, Denmark), 40% ethanol
(96%), 10% Acetic acid (Merck, Copenhagen, Denmark)
and 49.7% ddH2O. Gels were then destained in ddH2O
overnight and imaged using a ChemiDoc MP Imaging
System (Bio-Rad Laboratories). These Coomassie-stained
posttransferred gels were used to determine the amount of
protein in each lane, based on the MHC (~200 kDa)
bands (Murphy et al. 2006). Membranes were blocked in
Tris-buffered saline including 0.1% Tween-20 (TBST) with
either 2% skimmed milk or 3% BSA for 1 h and then
incubated with primary antibodies over night. After 2
washes in TBST, horseradish peroxidase-conjugated sec-
ondary antibody (DAKO, Glostrup, Denmark) diluted 1
to 5000 in TBST with addition of either 2% skimmed milk
or 3% BSA was added, following which the membranes
were washed in TBST (3 9 15 min). Bands were visual-
ized using chemiluminescent detection (single ﬁber analy-
sis using Super Signal West Femto Maximum Sensitivity
Substrate, Thermo Scientiﬁc, – muscle lysates using ECL,
Millipore) and images were collected on a ChemiDoc MP
Imaging System. For further analyses, the membrane was
kept in TBST and re-incubated in a new primary antibody
overnight, giving the opportunity to determine the expres-
sion of several proteins with different molecular weights
on the same segment of ﬁbers (Thomassen et al. 2013).
The membranes for single ﬁber analyses were divided
into four pieces by cutting over 250 kDa, right below the
150 kDa and 75 kDa, above the 25 kDa and below the
10 kDa markers (All Blue and Dual Color, Bio-Rad Labo-
ratories). The ﬁrst and upper part was used to conﬁrm the
predetermined ﬁber type (ST and FT 200 kDa), the second
part used for PFK (85 kDa) and SERCA1 (100 kDa), the
third for CS (48 kDa) and Actin (42 kDa), and the fourth
and lower part used for COX-4 (14 kDa).
Antibody details: Other antibodies used for protein
expression determination were: PFK: 0.2 lg∙mL1, mouse
monoclonal, Sc166722; Santa Cruz Biotechnology, Dallas,
TX;
CS:
0.33 lg∙mL1,
rabbit
polyclonal,
ab96600;
Abcam, Cambridge, UK; COX-4: 0.2 lg∙mL1, mouse
monoclonal, Sc58648; Santa Cruz Biotechnology.
Data treatment
In total, 160 segments of human skeletal muscle single
ﬁbers from vastus lateralis were used for the ﬁnal protein
expression determination in ST and FT ﬁbers. On each
gel 5 ST and 5 FT ﬁbers from a resting pre-HIT muscle
and 5 ST and 5 FT ﬁbers from a resting post-HIT muscle
from the same individual were loaded. Given the small
size of segments of individual ﬁbers, it was not possible
also to determine the total protein concentration in each
sample prior to sample loading. Consequently, different
amounts of protein were loaded in each well. In order to
compare the speciﬁc protein expression between ﬁber
samples, MHC on the post-transferred gel was quantiﬁed
and it was deemed that an equal proportion of total pro-
tein was transferred to the membrane independent of
total amount loaded. Thus, the signal on the posttrans-
ferred Coomassie stained gel was used for normalization
of the densities of the protein of interest, as previously
demonstrated as a reliable measure (Murphy et al. 2006;
Thomassen et al. 2013).
The signal intensity for each protein of interest was ﬁrst
normalized to the mean intensity of all single human ﬁber
bands for that protein on the gel. Afterward data were
normalized to the total amount of protein in each sample
determined by Coomassie staining of the remaining MHC.
In order to compare ﬁbers loaded on different gels single
values were normalized to the mean of ST pre-HIT in the
single ﬁber analysis and pre-HIT in the muscle homoge-
nate analysis. Finally, in order to have a normal distribu-
tion of the data, the ratios were log transformed before the
statistical analysis. For clarity the graphical presentation of
the results are displayed as ratios relative to ST pre-HIT
for single ﬁber data and from the backtransformed log val-
ues relative to pre-HIT for muscle homogenate.
Capillary density
Capillarization was analyzed using ﬂuorescence micros-
copy. Transverse sections of the muscle biopsies were cut
2015 | Vol. 3 | Iss. 7 | e12428
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ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Metabolic and Muscle Adaptations to HIT
P. M. Christensen et al.
at a thickness of 8 lm and placed onto glass slides and
ﬁrst treated with Biotinylated Ulex Europaeus Agglutinin
I (Vector Laboratories, Burlingame, CA) and later with
Streptavidin 1:200 (Dako, Glostrup, Denmark). Subse-
quently pictures were taken of the muscle samples and
analyzed on a computer (ImageJ) for capillary to ﬁber
ratio.
Statistics
Before and after HIT changes in _VO2 kinetics, capillary to
ﬁber ratio, muscle metabolites at rest and after 3 and
6 min of exercise as well as enzyme activity and protein
level in homogenates were examined with a Student’s
paired t-test. Also a paired t-test was used to evaluate if s
of the primary phase differed between MOD and INT
both pre- and post-HIT. Changes in segments of single
muscle ﬁbers were evaluated using a two-way ANOVA
General Linear Model (ST vs. FT ﬁbers and pre vs. post-
HIT as factors). The ﬁve ST ﬁbers were averaged to yield
one value for each subject both pre- and post-HIT and
the same approach was made for the ﬁve FT ﬁbers. If sig-
niﬁcant main effects or an interaction were observed, then
a student Newman-Keuls post hoc analysis was performed
to identify the speciﬁc differences in protein expression
within ﬁber types. Correlations between _VO2 kinetics (s
of the primary response during MOD and INT and the
relative VO2 slow component) and maximal aerobic
enzyme activity and the capillary to ﬁber ratio was evalu-
ated using a one-tailed test with the a-priory hypothesis
that fast _VO2 kinetics and a minor _VO2 slow component
would be associated with a high aerobic enzyme activity
and capillary to ﬁber ratio.
Results
Muscle adaptations
After HIT no overall effect of the intervention was
observed for the amount of CS (P = 0.12; Fig. 2A), COX-
4 (P = 0.20; Fig. 2B) and PFK (P = 0.70; Fig. 2C) in seg-
ments of ST and FT ﬁbers. An overall effect was found
for ﬁber type for CS (P = 0.01), COX-4 (P = 0.007) and
PFK (P < 0.001) since protein content in ST ﬁbers was
higher than FT ﬁbers for CS (P = 0.016 pre-HIT and
P = 0.007 post-HIT) and COX-4 (P = 0.012 pre-HIT and
P = 0.01 post-HIT) and lower for PFK (P < 0.001 pre
and post-HIT). Before HIT the content in ST ﬁbers of CS
and COX-4 was on average 53% and 41% higher than in
FT ﬁbers whereas PFK was ~389% higher in FT ﬁbers
with respective values after HIT being 34%, 15% and
~241%. In muscle
homogenates
both
CS (P = 0.07;
Fig. 2A) and COX-4 (P = 0.10; Fig. 2B) tended to be
lower after compared to before HIT with an average
decrease in protein content of 16% and 11%, respectively.
PFK remained unchanged (P = 0.45; Fig. 2C) with an
average decrease of 3%. Representative Western blots of
the proteins investigated are displayed in Fig. 3.
After HIT the maximal enzyme activity was not changed
relative to before the intervention for CS (56  8 vs.
59  10 lmol g DW1 min1; P = 0.10), HAD (27  6
vs. 29  3 lmol g DW1 min1; P = 0.41), LDH (131 
27 vs. 113  27 lmol g DW1 min1; P = 0.14) and PFK
(340  69 vs. 318  105 lmol g DW1 min1; P = 0.49).
Following HIT no changes relative to before the inter-
vention were observed in the capillary to ﬁber ratio
(2.30  0.16 vs. 2.38  0.20; P = 0.13) (Fig. 4A).
_VO2 kinetics
No signiﬁcant differences following HIT were observed in
the _VO2 response during MOD (Fig. 5) and in all modeling
parameters (Table 1) as s was unchanged (P = 0.67)
together with absolute _VO2 (P = 0.39), RER (P = 0.63)
and GE (P = 0.39) averaged over the last 2 min of exercise.
The _VO2 response during INT was also unaffected by
HIT (Fig. 5) as were all modeling parameters (Table 1)
including s of the primary response (P = 0.32), the abso-
lute (P = 0.26) and relative (P = 0.25) size of the _VO2
slow component of the secondary response together with
absolute
_VO2
(P = 0.52),
RER
(P = 0.13)
and
GE
(P = 0.46) averaged over the last 2 min of exercise.
s during MOD and INT was not different neither pre
(P = 0.12) nor post-HIT (P = 0.22).
Correlations
Maximal activity of CS (r2 = 0.002–0.18; P > 0.05) and
HAD (r2 = 0.0001–0.16; P > 0.05) did not correlate with s
of the primary response during MOD and INT both before
and after HIT. Neither did CS correlate with the relative
size of the _VO2 slow component (r2 = 0.38 and 0.30 before
and after HIT; P > 0.05) as was the case for HAD
(r2 = 0.12 and 0.30 before and after HIT; P > 0.05). The
capillary to ﬁber ratio was associated with s during MOD
before (r2 = 0.90; P < 0.001) but not after HIT (r2 = 0.03;
P > 0.05) and no association was present during INT
(r2 = 0.10–0.14; P > 0.05). An association between the cap-
illary to ﬁber ratio and the relative size of the _VO2 slow
component (Fig. 4B) was present following HIT (r2 = 0.39;
P < 0.05) but not before HIT (r2 = 0.38; P > 0.05).
Muscle metabolites
Muscle CP (n = 7) was not changed by HIT at rest
(~90 mmol g DW1 min1; P = 0.60) and after 3 min of
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 7 | e12428
Page 7
P. M. Christensen et al.
Metabolic and Muscle Adaptations to HIT
INT (~60 mmol g DW1 min1; P = 0.41), but after HIT
it was lower at 6 min of exercise than before HIT (42 
15 vs. 66  17 mmol kg DW1; P = 0.011) (Fig. 6A).
Muscle lactate (n = 7) was not changed by HIT at rest
(~7 mmol g DW1 min1; P = 0.18) and after 3 min of
INT (~17 mmol g DW1 min1; P = 0.95), but at 6 min
muscle lactate was higher after compared to before HIT
(40.2  18.4 vs. 14.1  5.5 mmol kg DW1; P < 0.012)
(Fig. 6B).
Discussion
The major ﬁndings in the present study were that a per-
iod of reduced and intensiﬁed training performed by
trained cyclists did not elevate the protein content of oxi-
dative enzymes in FT ﬁbers. Furthermore, no components
of pulmonary _VO2 kinetics were changed, whereas higher
muscle lactate accumulation and lower level of CP were
observed during intense cycling after compared to before
the intervention period.
Contrary to our hypothesis the content of CS and
COX-4 in the FT ﬁbers was not elevated after the inter-
vention period in the form of volume reduced and inten-
siﬁed training. The high exercise intensity used in HIT, in
particular the repeated sprint training, was chosen in
order to activate all FT ﬁbers with this type of exercise
being a potent stimulator of the signal cascades leading to
adaptations of the muscular oxidative system (Psilander
0
0,5
1
1,5
2
1.5
0
2
4
6
8
10
0,5
1
1,5
0
0,5
1
HOM
COX-4 signal intensity
(relative to PRE)
0
1
1,5
PFK signal intensity
(relative to PRE)
0
0,5
1
CS signal intensity
(relative to PRE)
1.5
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.5
A
B
C
# 
CS signal intensity
(relative to ST PRE)
COX-4 signal intensity
(relative to ST PRE)
0.5
0
1.0
PFK signal intensity
(relative to ST PRE)
ST PRE                        ST POST                        FT PRE                          FT POST   
HOM
# # 
# # # 
0.5
2.7
0
1.0
0
100
200
300
400
500
PRE
POST
PFK maximal activity
(µmol/g DW/min)
(µmol/g DW/min)
0
20
40
60
80
CS maximal activity
D
E
F
H
G
Figure 2. Enzyme content following 7 weeks of high intensity and reduced volume training in trained cyclists (n = 8). The average content
(thick lines) of CS (A), COX-4 (B), and PFK (C) before (PRE) and after (POST) the intervention are shown in slow-twitch (ST) and fast-twitch (FT)
ﬁbers together with individual values (average of ﬁve ﬁbers  SEM at each time point) which have been normalized to ST PRE for all enzymes.
Average and individual protein content measured in homogenates (HOM) are also shown for CS (D), COX-4 (E), and PFK (F) as well as maximal
activity for CS (G) and PFK (H). For clarity the graphs A-F display ratio data but the statistical analysis was based on log data. #P < 0.05,
##P < 0.01, ###P < 0.001; signiﬁcant difference between ST and FT ﬁbers.
2015 | Vol. 3 | Iss. 7 | e12428
Page 8
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Metabolic and Muscle Adaptations to HIT
P. M. Christensen et al.
et al. 2010; Little et al. 2011). Thus, it was unexpected
that no change in oxidative enzyme content was present
in FT ﬁbers after the training period. One explanation
may be that the cyclists during their normal training and
competition prior to the intervention period, routinely
engaged most of their muscle ﬁbers, and that “extra” FT
ﬁbers activated during the training in the intervention
period were only ﬁbers from the highest order motor
units. Then, the FT ﬁbers in “lower” order motor units
were recruited less due to the reduction in training vol-
ume from ~470 to ~240 min week1. In support of this,
it has been shown that, even at 65% (Scribbans et al.
2014) and 80% (Krustrup et al. 2004b) of _VO2-max, a
signiﬁcant number of FT ﬁbers are recruited. In the pres-
ent study a considerable part of the training prior to
HIT was performed near 80% _VO2-max (~28% of total
0
5
10
15
20
25
30
Relative VO2 slow component 
(% of end exercise VO2) 
Capillary to fibre ratio
PRE
POST
Capillary to fibre ratio
1.9
2.1
2.3
2.5
2.7
1.9
2.1
2.3
2.5
2.7
A
B
Figure 4. Muscle capillary to ﬁber ratio (A) before (PRE; open bars) and after (POST; closed bars) 7 weeks of high intensity and reduced
volume training in trained cyclists (n = 8) with insert picture showing staining of capillaries in a representative subject. Values are means  SD.
Association between the capillary to ﬁber ratio and the relative _VO2 slow component (B) before (open symbols, r2 = 0.38; P > 0.05) and after
(closed symbols, r2 = 0.39; P < 0.05) 7 weeks of high intensity and reduced volume training in trained cyclists (n = 8).
MHCI
200 kDa
PRE
POST
ST
ST
FT
FT
200 kDa
MHC Coomassie
CS
200 kDa
MHCII
SERCA1
PFK
COX-4
100 kDa
85 kDa
48 kDa
14 kDa
14 kDa
85 kDa
48 kDa
CS
PFK
COX-4
PRE
POST
HOM
Figure 3. Representative western blots of the proteins investigated in slow-twitch (ST), fast-twitch (FT), and muscle homogenate (HOM) before
(PRE) and after (POST) 7 weeks of high intensity and reduced volume training in trained cyclists. See methods section for details.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 7 | e12428
Page 9
P. M. Christensen et al.
Metabolic and Muscle Adaptations to HIT
training time or ~130 min week1 was carried out with a
heart rate above 80% of HR-max). Furthermore, detrain-
ing in trained athletes is known to reduce content and
maximal activity of oxidative enzymes (Chi et al. 1983;
Christensen et al. 2011). Thus, it may be that the protein
content of oxidative enzymes was reduced in the motor
units controlling FT ﬁbers lower in the ﬁber hierarchy
due to the reduction in training. This may also explain
the overall tendency for a drop in protein content and
maximal activity of CS and COX-4 measured in muscle
homogenates.
The single ﬁber data of CS and COX-4 showed a large
variation in oxidative enzyme protein content within each
ﬁber type as evidenced by the high standard error for
many of the cyclists (Fig. 2A and B). This is in agreement
with the pioneering work by Lowry et al. (1978) showing
a large range in maximal enzyme activity in single ﬁbers,
but in that study a separation between ST and FT ﬁbers
was not made. The observed difference in the content of
muscle oxidative enzymes between individual ﬁbers sup-
ports the proposed concept that motor units have a range
from very fast to very slow _VO2 kinetics as well as differ-
ent steady-state values (Koppo et al. 2004). Studies using
trained subjects (Jansson and Kaijser 1977; Chi et al.
1983) and a longitudinal study on untrained subjects
(Henriksson and Reitman 1976) have shown that the
maximal activity of oxidative enzymes in FT ﬁbers can be
as high as in ST ﬁbers. This was not the case in the pres-
ent study, which may be due to a higher aerobic training
status ( _VO2-max: ~70 mL min1 kg1) of the subjects in
the studies showing similar levels in FT and ST ﬁbers
(Jansson and Kaijser 1977; Chi et al. 1983). Nevertheless,
the
combination
of
repeated
30-sec
sprinting
(2.5 9 week1)
and
aerobic
high
intensity
training
(1.5 9 week1) in the present study with a reduced train-
ing volume did not increase the oxidative enzyme content
in FT ﬁbers in already trained athletes with a _VO2-max of
~60 mL min1 kg1. On the other hand, it has been
Table 1. Pulmonary
_VO2 kinetics modeling parameters during
moderate (MOD) and intense (INT) cycling before (PRE) and after
(POST) 7 weeks of high intensity and reduced volume training in
trained cyclists (n = 8). Changes in pre and post were evaluated
with a paired t-test.
PRE
POST
MOD
Baseline (mL min1)
865  96
849  60
TdP (sec)
17.5  3.1
18.3  2.9
sP (sec)
15.9  2.4
15.4  2.5
AP (mL min1)
2209  119
2249  144
_VO2 4–6 min (mL min1)
3083  172
3110  165
RER 4–6 min
0.95  0.02
0.95  0.01
GE 4–6 min (%)
19.1  0.7
18.9  0.7
Cadence (rounds min1)
92  9
92  8
INT
Baseline (mL min1)
870  65
824  53
TdP (sec)
15.2  1.5
15.3  2.3
sP (sec)
18.2  2.5
17.2  2.9
AP (mL min1)
2931  240
2990  186
TdS (sec)
103  30
95  56
sS (sec)
141  60
181  111
AS (mL min1)
479  264
539  257
_VO2 4–6 min (mL min1)
4153  234
4180  184
RER 4–6 min
1.03  0.04
1.01  0.02
GE 4–6 min (%)
19.6  0.9
19.5  0.7
Cadence (rounds min1)
93  6
94  6
Values are means  SD. Baseline ( _VO2 before onset of exercise).
Time delay (Td), time constant (s), amplitude (A) for the primary
response (P), and the slow component (S). Absolute oxygen uptake
( _VO2), respiratory exchange ratio (RER), and gross efﬁciency (GE)
in the last 2 min of exercise from 4–6 min.
0
1500
3000
4500
–120 –60
0
60
120 180 240 300 360
Time (sec)
0
1500
3000
4500
–120 –60
0
60
120 180 240 300 360
VO2
(mL/min) 
Time (sec)
A
B
0
1500
3000
4500
–120 –60
0
60
120 180 240 300 360
Time (sec)
C
Figure 5. Pulmonary oxygen uptake ( _VO2) following 7 weeks of high intensity and reduced volume training in trained cyclists (n = 8) during
moderate (MOD; A) and intense (INT; B) cycling with the modeled responses shown (C) before (PRE; open symbols and hatched lines) and after
(POST; ﬁlled symbols and solid lines) the intervention. The arrow in panel C indicates the onset of the _VO2 slow component during INT (103
and 95 sec on average PRE and POST HIT) being superimposed on the primary _VO2 response.
2015 | Vol. 3 | Iss. 7 | e12428
Page 10
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Metabolic and Muscle Adaptations to HIT
P. M. Christensen et al.
shown that adding two weekly aerobic high intensity ses-
sions (6 9 ~3 min with ~1.5 min recovery), at the speed
eliciting task failure in an incremental test, increased the
content of LDH in the FT ﬁbers after 6 weeks of training
with a maintained training volume in well-trained run-
ners ( _VO2-max: 67 mL min1 kg1) (Kohn et al. 2011).
Therefore, it seems plausible that the training reduction
in the present study can explain the lack of increase in
oxidative protein content in FT ﬁbers. Alternatively, train-
ing with repeated sprints may be less potent than aerobic
high intensity training (3–5 min intervals at ~90–100%
_VO2-max)
to
increase
protein
content
of
oxidative
enzymes in FT ﬁbers in trained individuals, since the for-
mer type of training dominated in the present study.
Based on changes in blood and muscle lactate in
trained individuals it does appear that different types of
high intensity training can yield different outcomes.
Accordingly, aerobic high intensity training (< _VO2-max;
e.g., 3–5 min intervals) and a maintained training volume
appears to lower muscle (Clark et al. 2004) and blood
lactate (Acevedo and Goldfarb 1989; Kohn et al. 2011)
during intense exercise (< _VO2-max), whereas training
interventions with repeated 30-sec sprints in combination
with a volume reduction does not lower blood lactate
(Bangsbo et al. 2009; Iaia et al. 2009) and in the present
study muscle lactate was higher after training. Future
studies are needed to evaluate if the outcome on oxidative
adaptations in FT ﬁbers differs between different types of
high intensity training which appears to be the case with
regards to lactate levels. The role of the total training vol-
ume is considered of interest, thus either adding SET on
top of the normal training or having SET substitute less
intense training seems relevant.
Pulmonary _VO2 kinetics was not changed with HIT
(Fig. 5 and Table 1). This ﬁnding differs from the faster
_VO2 kinetics observed after a period of intense training
in untrained subjects at both the muscular (Krustrup
et al. 2004a) and pulmonary level (Bailey et al. 2009) with
the latter study also reporting a reduced _VO2 slow com-
ponent. Despite the tendencies for a reduction in the con-
tent of CS and COX-4 as well as the maximal activity of
CS
in
the
present
study,
_VO2
kinetics
remained
unchanged. These ﬁndings suggest that the level of oxida-
tive enzymes does not limit the muscle oxygen utilization
in the initial part of exercise, nor the development of the
_VO2 slow component. It should, however, be considered
that the content and maximal activity of the oxidative
enzymes was not signiﬁcantly reduced, and it may be that
endurance athletes have an excessive oxidative enzyme
capacity allowing a modest drop without having an effect
on _VO2 kinetics. The CS activity measured in muscle
homogenate was ~60 lmol g DW1 min1, which is
about twice as high as in untrained subjects (Krustrup
et al. 2004a; Burgomaster et al. 2005, 2008) and some-
what higher than previous observations in trained endur-
ance athletes with a similar _VO2-max as in the present
study (Yeo et al. 2008; Bangsbo et al. 2009). In a study of
trained soccer players 2 weeks without training resulted
in a decrease in activity and content of oxidative enzymes,
which was associated with slower _VO2 kinetics (Christen-
sen et al. 2011). Thus, it cannot be excluded that the level
of oxidative enzymes under some circumstances may
become limiting for _VO2 kinetics in trained individuals.
Unlike previous ﬁndings in trained individuals (Koppo
et al. 2004) the speed of the primary _VO2 response was
not signiﬁcantly different between MOD (s~15.7 sec) and
INT (s~17.7 sec) although ﬁve of eight subjects had larger
s in INT than in MOD both pre- and post-HIT. Thus,
despite an expected larger recruitment of FT ﬁbers in INT
relative to MOD (Krustrup et al. 2004b) and the fact that
FT
ﬁbers
had
markedly
lower
content
of
oxidative
enzymes than ST ﬁbers in the present study (Fig. 2A and
B), the _VO2 kinetics of the primary response in INT was
not slower than MOD. This in turn suggests that at least
in ST ﬁbers there is an excess capacity of oxidative
enzymes that does not impact on the speed of the VO2
response. Maximal oxidative enzyme activity of CS and
HAD were poor predictors of fast _VO2 kinetics and the
relative size of the _VO2 slow component showing that in
trained individuals these enzymes appear to be of minor
importance and other muscular variables needs to investi-
gated.
Of
interest
was
the
association
between
the
0
10
20
30
40
50
60
70
Muscle lactate
(mmol/kg DW)
0
20
40
60
80
100
120
Creatine phosphate
(mmol/kg DW)
A
B
*
*
PRE      POST
Rest
PRE      POST
3 min
PRE       POST
6 min
Figure 6. Muscle creatine phosphate (A) and lactate (B) at rest and
after 3 and 6 min of intense cycling before (PRE; open bars) and
after (POST; closed bars) 7 weeks of high intensity and reduced
volume training in trained cyclists (n = 7). Values are meansSD.
*P < 0.05; signiﬁcant difference between PRE- and POST-values.
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2015 | Vol. 3 | Iss. 7 | e12428
Page 11
P. M. Christensen et al.
Metabolic and Muscle Adaptations to HIT
capillary to ﬁber ratio and the relative size of the _VO2
slow component after the HIT intervention and the
marked tendency for an association before HIT (Fig. 4).
However, the association was mainly due to one subject
having a low capillary to ﬁber ratio. Nevertheless, during
intense exercise eliciting a slow component – likely due to
recruitment of FT ﬁbers (Jones et al. 2011) – a high capil-
lary to ﬁber ratio could be speculated to lower blood
mean transit time optimizing conditions for diffusion of
oxygen which is supported by the ﬁnding of a reduced
_VO2 slow component during exercise inhaling an hyper-
oxic gas (Wilkerson et al. 2006).
The decrease in muscle CP and the increase in mus-
cle lactate from 3 to 6 min in INT (~85% _VO2-max)
were larger after the HIT period (Fig. 6). These ﬁndings
suggest a larger anaerobic energy turnover in the last
phase of INT. The activity of PFK was not elevated
with the intervention period, neither when expressed as
maximal activity or content, so it cannot explain the
apparently greater rate of glycolysis (Spriet et al. 2000).
The larger drop in CP indicates a higher accumulation
of muscle ADP, which may have elevated the rate of
glycolysis leading to a greater lactate production, but a
larger ADP concentration would also be expected to
stimulate the respiration but that was not the case in
light of the unchanged _VO2 response. Alternatively, the
tendency for a lower content and maximal activity of
muscle oxidative enzymes (CS and COX-4) after the
HIT period may have reduced the mitochondrial utili-
zation of the produced pyruvate (Brooks 2000), thereby
enhancing
the
lactate
production
catalyzed
by LDH
(Spriet et al. 2000). It is, however, unclear, why such
changes did not occur in the ﬁrst phase (0–3 min) of
INT (Fig. 6B). Nevertheless, it appears that the anaero-
bic energy ﬂux during the last phase (3–6 min) of INT
was higher after the intervention period, and thus, total
energy
turnover,
as
pulmonary
VO2
was
unaltered.
Alternatively, this ﬁnding may reﬂect a greater imbal-
ance between muscle CP and creatine and a change in
the ratio between muscle lactate and pyruvate during
exercise due to altered regulation. In moderately trained
subjects (VO2-max: 49 mL min1 kg1) repeated sprint
training, as used in the present study, resulted in lower
muscle lactate and ATP concentrations after exercise at
an intensity corresponding to 90% of _VO2-max. How-
ever, there were major differences between the present
study and the one by Burgomaster and co-workers
including a lower training status, and an increase in
training volume and a higher maximal aerobic enzyme
activity after the training period (Burgomaster et al.
2006) which may in part explain the different change
in muscle lactate accumulation during intense exercise.
The ﬁndings in the present study are in contrast to
observations in a study also using well-trained cyclists
( _VO2-max: ~65 mL O2 min1 kg1) who for a 3-week
period added aerobic high intensity training (8 9 5 min
~85%
_VO2-max) three times weekly to their normal
training volume. Following the training period muscle
lactate
accumulation
during
intense
exercise
(~85%
_VO2-max)
was
reduced
and
during
more
moderate
exercise (65% _VO2-max) fat and carbohydrate oxidation
was larger and lower, respectively (Clark et al. 2004).
Content
and
maximal
activity
of
oxidative
enzymes
were
not
reported,
but
was
unchanged
in
trained
cyclists in another study using the same type of training
(Yeo et al. 2008). Taken together these ﬁndings suggest
that the reduced amount of training in the present
study is the major cause of the elevated anaerobic
energy production during the intense submaximal work.
Furthermore, the present study shows that anaerobic
metabolism can be altered without a change in
_VO2
kinetics. Such dissociation between anaerobic and aero-
bic metabolism has also been reported in the exercise
transient in studies using hyperoxia where CP utiliza-
tion has been observed to be reduced (Vanhatalo et al.
2010) and the primary
_VO2 response appears to be
unaffected
(Wilkerson
et al.
2006).
Likewise,
during
steady-state conditions with moderate exercise higher
CP and lower muscle lactate have been observed in hy-
peroxia
(Stellingwerff
et al.
2006)
despite
the
_VO2
response being similar in both the exercise transient
and in the stable phase of exercise (Wilkerson et al.
2006). Further studies are needed to evaluate how dif-
ferent training regimes inﬂuence anaerobic energy turn-
over during submaximal exercise.
The functional signiﬁcance of the training period in the
present study has been reported previously with regard to
high intensity
exercise
performance
in
the
form
of
improved
performance
in
a
repeated
sprint
test
(6 9 20 sec) and in an exhaustive test lasting ~4 min
with the latter test being preceded by a 2-min preload
with high intensity in which muscle lactate at the end of
the preload also was elevated after HIT (Gunnarsson et al.
2013). This indicates that the apparently larger anaerobic
muscle perturbation with HIT does not lower perfor-
mance during intense exercise.
Each subject had ﬁve ST and FT ﬁbers analyzed both
pre and post-HIT (total of 20 ﬁbers) and the average
value of the ﬁve ﬁbers for each time point (pre- and
post-HIT) was used for further analysis. Such a low num-
ber may seem limiting, but a statistical difference was
present between ST and FT ﬁbers for all enzymes investi-
gated in line with previous reports using pooled groups
of ST and FT ﬁbers to determine maximal enzyme activ-
ity (Essen et al. 1975; Essen-Gustavsson and Henriksson
1984; Schantz and Henriksson 1987) or content (Thomas-
2015 | Vol. 3 | Iss. 7 | e12428
Page 12
ª 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Metabolic and Muscle Adaptations to HIT
P. M. Christensen et al.
sen et al. 2013). Furthermore, the tendencies for a drop
in homogenate protein content and maximal activity for
CS and COX-4 and unchanged PFK levels were mirrored
by the single ﬁber data. In addition the same three sub-
jects who had a higher CS content in FT ﬁbers after HIT
also had elevated content of COX-4 (Symbols □, ■ and
▲on Fig. 2). Taken together this suggests that the method
is sensitive enough to detect possible changes in response
to a training intervention.
In
summary,
the
present
study
showed
that
the
amount of CS and COX-4 in FT ﬁbers and muscle
homogenate as well as maximal activity of CS was not
changed after a 7-week period of intensiﬁed training with
a reduced volume in already trained cyclists. Further-
more,
no
change
in
pulmonary
_VO2
kinetics
was
observed during moderate (~65% _VO2-max) and intense
(~85% _VO2-max) submaximal exercise. During intense
cycling muscle CP levels were reduced and muscle lactate
accumulation was elevated to a greater extent in the later
part of exercise (3–6 min) following the training inter-
vention without an altered
_VO2. This could be inter-
preted to reﬂect a larger total energy turnover following
the training intervention due to a greater anaerobic
energy contribution to exercise or an altered regulation
of anaerobic muscle metabolism.
Acknowledgments
Lars Ropke, Rasmus Vilsgaard, Lars Nybo, Nikolai Nords-
borg for assistance during the experiments, Rafael Casuso
and Karina Olsen for assistance with muscle analysis and
the subjects for their great effort and determination dur-
ing training and testing.
Conﬂict of Interest
No conﬂict of interest is reported.
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Unchanged content of oxidative enzymes in fast-twitch muscle fibers and V˙O2 kinetics after intensified training in trained cyclists.
Christensen, Peter M,Gunnarsson, Thomas P,Thomassen, Martin,Wilkerson, Daryl P,Nielsen, Jens Jung,Bangsbo, Jens