ORIGINAL RESEARCH
Effect of speed endurance training and reduced training
volume on running economy and single muscle ﬁber
adaptations in trained runners
Casper Skovgaard1,2, Danny Christiansen3, Peter M. Christensen1,2, Nicki W. Almquist1,
Martin Thomassen1 & Jens Bangsbo1
1 Department of Nutrition, Exercise and Sports, Section of Integrative Physiology, University of Copenhagen, Copenhagen, Denmark
2 Team Danmark (Danish Elite Sports Organization), Copenhagen, Denmark
3 Institute of Sport, Exercise and Active Living (ISEAL), Victoria University, Melbourne, Australia
Keywords
Intense training, muscle ﬁber type-speciﬁc
adaptations, muscular adaptations, sprint
interval training.
Correspondence
Jens Bangsbo, University of Copenhagen,
Department of Nutrition, Exercise and Sports,
Section of Integrative Physiology, August
Krogh Building, Universitetsparken 13, 2100
Copenhagen O, 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 a grant from
Team Danmark (Danish elite sports
organization), Copenhagen, Denmark.
Received: 26 October 2017; Revised: 7
January 2018; Accepted: 9 January 2018
doi: 10.14814/phy2.13601
Physiol Rep, 6 (3), 2018, e13601,
https://doi.org/10.14814/phy2.13601
Abstract
The aim of the present study was to examine whether improved running
economy with a period of speed endurance training and reduced training vol-
ume could be related to adaptations in speciﬁc muscle ﬁbers. Twenty trained
male (n = 14) and female (n = 6) runners (maximum oxygen consumption
(VO2-max): 56.4  4.6 mL/min/kg) completed a 40-day intervention with 10
sessions of speed endurance training (5–10 9 30-sec maximal running) and a
reduced (36%) volume of training. Before and after the intervention, a muscle
biopsy was obtained at rest, and an incremental running test to exhaustion
was performed. In addition, running at 60% vVO2-max, and a 10-km run was
performed in a normal and a muscle slow twitch (ST) glycogen-depleted con-
dition. After compared to before the intervention, expression of mitochondrial
uncoupling protein 3 (UCP3) was lower (P < 0.05) and dystrophin was higher
(P < 0.05) in ST muscle ﬁbers, and sarcoplasmic reticulum calcium ATPase 1
(SERCA1) was lower (P < 0.05) in fast twitch muscle ﬁbers. Running econ-
omy at 60% vVO2-max (11.6  0.2 km/h) and at v10-km (13.7  0.3 km/h)
was ~2% better (P < 0.05) after the intervention in the normal condition, but
unchanged in the ST glycogen-depleted condition. Ten kilometer performance
was improved (P < 0.01) by 3.2% (43.7  1.0 vs. 45.2  1.2 min) and 3.9%
(45.8  1.2 vs. 47.7  1.3 min) in the normal and the ST glycogen-depleted
condition, respectively. VO2-max was the same, but vVO2-max was 2.0%
higher (P < 0.05; 19.3  0.3 vs. 18.9  0.3 km/h) after than before the inter-
vention. Thus, improved running economy with intense training may be
related to changes in expression of proteins linked to energy consuming pro-
cesses in primarily ST muscle ﬁbers.
Introduction
Speed endurance training (SET; 10–40 sec repeated “all-
out” efforts with rest periods lasting >5 times the exercise
bouts) with a concomitant reduced training volume has
been found to improve endurance performance in associ-
ation with better running economy at submaximal speeds
in trained runners (Bangsbo et al. 2009; Bangsbo 2015).
However, the mechanisms causing the improved running
economy are not clearly identiﬁed, but may be related to
metabolic changes in the trained muscles (Saunders et al.
2004).
Training-induced improvement in running economy
may be due to higher mitochondrial efﬁciency, that is,
higher ATP/O2, which could be due to reduced uncou-
pled respiration. The mitochondrial uncoupling protein 3
ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
2018 | Vol. 6 | Iss. 3 | e13601
Page 1
Physiological Reports ISSN 2051-817X
(UCP3) is suggested to be involved in thermogenesis by
dispersing energy as heat instead of converting it to ATP
(Gong et al. 1997; Boss et al. 2000) and improved run-
ning economy may therefore be related to reduced levels
of muscle UCP3. In agreement, cross-sectional studies
have shown that endurance-trained subjects have lower
muscle UCP3 expression and better running economy
than untrained subjects (Russell et al. 2003a,b; Mogensen
et al. 2006). However, Iaia et al. (2009) found no change
in whole muscle UCP3 level although running economy
improved after 4 weeks of SET and a 65% reduced train-
ing volume. Thus, studies should investigate whether
changes in the single muscle ﬁber expression of UCP3
could be related to changes in running economy.
The transfer of muscle force produced by the acto-
myosins involves a secondary matrix of proteins that trans-
mit the muscle force along and between muscle ﬁbers and
out to the extracellular matrix. Cytoskeleton proteins, such
as dystrophin, have been identiﬁed as playing a role in this
muscle force transmission (Rybakova et al. 2000; Prins
et al. 2009) and changes in the expression of these proteins
could inﬂuence the integrity and the strength of the muscle
(Hughes et al. 2015). Hence, increased expression of mus-
cle dystrophin may result in increased rate of force devel-
opment, increased muscular power output and greater
storage and return of elastic energy thereby lowering the
cost of running (i.e., improve running economy).
Another potential cause of training-induced improve-
ments in running economy is lowered muscle expression
of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)
pumps, as they are suggested to be responsible for up to
50% of the ATP used during muscle activity (Clausen
et al. 1991; Walsh et al. 2006; Smith et al. 2013). Studies
have shown that speed endurance training modulates
skeletal muscle ﬁber type distribution in soccer players
(Gunnarsson et al. 2012) and runners (Skovgaard et al.
2014), which has been found together with lowered
SERCA1 expression (Skovgaard et al. 2014) and improved
running economy. Muscle ﬁbers with high SERCA1
expression have a faster release and uptake of Ca2+ (Del-
bono and Meissner 1996; Froemming et al. 2000) and
lowered expression of SERCA1 may therefore reduce the
energy turnover during exercise.
An increase in the respiratory capacity of skeletal mus-
cle permits the use of less oxygen per mitochondrial res-
piratory chain for a given submaximal running speed
(Saunders et al. 2004). Slow twitch (ST) muscle ﬁbers
have higher mitochondrial content and are more depen-
dent on oxidative metabolism than fast twitch (FT) mus-
cle ﬁbers (Berchtold et al. 2000; Schiafﬁno and Reggiani
2011). However, Jansson and Kaijser (1977) reported that,
unlike a control group of varying physical ﬁtness, there
was no difference in succinate dehydrogenase muscle
activity between ST and FT ﬁbers in gastrocnemius mus-
cle of elite orienteers, suggesting that FT ﬁbers have the
ability to metabolically adapt to high oxidative demands
(Jansson and Kaijser 1977). Metabolic adaptations in FT
ﬁbers may therefore contribute to improving running
economy after intense training, such as SET, targeting
both ST and FT ﬁbers (Egan and Zierath 2013). In sup-
port, augmented mRNA response related to mitochon-
drial
biogenesis
(peroxisome
proliferator-activated
receptor-c coactivator-1, PGC-1a) and metabolism (hex-
okinase II and pyruvate dehydrogenase kinase-4, PDK4)
in trained subjects was observed following a SET session
(Skovgaard et al. 2016). Furthermore, PGC-1a mRNA has
been shown to increase in an exercise intensity-dependent
manner (Egan et al. 2010; Nordsborg et al. 2010). Regular
intense training may therefore lead to higher oxidative
capacity, possibly due to oxidative adaptations in FT
ﬁbers, which in turn could contribute to the improved
running economy as a result of the intense training (Iaia
et al. 2008; Bangsbo et al. 2009; Iaia and Bangsbo 2010;
Skovgaard et al. 2014).
In vitro studies have shown that the energy cost of con-
traction is higher in FT than ST ﬁbers (Crow and Kushmer-
ick 1982; Barclay et al. 1993; He et al. 2000). This was
conﬁrmed in vivo by Krustrup et al. (2008) who observed
that the oxygen uptake for at given exercise intensity was
higher when ST ﬁbers were blocked by a neuromuscular
blocking agent. And reports by Krustrup et al. (2004), who
depleted the ST ﬁbers the day before submaximal exercise,
that the glycogen depletion of ST ﬁbers enhanced the
recruitment of FT ﬁbers and elevated the energy require-
ment by 7% (Krustrup et al. 2004). By using the approach,
of depleting ST ﬁbers the day before exercise (Krustrup
et al. 2004), before and after a SET period, it may be possi-
ble to study whether a change in running economy is
caused by speciﬁc adaptations in FT ﬁbers.
Thus, the aims of the present study were in trained run-
ners to investigate the effect of intensiﬁed training, in the
form of speed endurance training and a reduced volume of
aerobic training, on running economy and adaptation of
single muscle ﬁbers. We hypothesized that FT muscle ﬁbers
would adapt to the training by lowered expression of
UCP3 and SERCA1, and increased expression of dys-
trophin and CS, which would be associated with improved
running economy and 10-km running performance.
Methods
Subjects
Twenty-six trained runners commenced the study. Six sub-
jects did not complete the intervention period due to per-
sonal circumstances (n = 4) or low adherence to the
2018 | Vol. 6 | Iss. 3 | e13601
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ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
C. Skovgaard et al.
training program (n = 2). Thus, a total of twenty trained
male (n = 14) and female (n = 6) runners with an average
age, height, body mass, and maximum oxygen consump-
tion
(VO2-max)
of
28.1  4.5 years,
177.5  9.9 cm,
72.5  10.6 kg, and 56.4  4.6 mL/min/kg, respectively,
(males: 28.8  4.8 years, 181.8  7.9 cm, 77.8  6.6 kg,
58.1  3.4 mL/min/kg;
females:
27.4  3.7 years,
169.0  5.6 cm,
59.9  6.9 kg,
52.5  4.9 mL/min/kg;
means  SD), completed the study. After receiving written
and oral information about the study and the possible risks
and discomforts associated with the experimental proce-
dures, all subjects gave their written informed consent to
participate. The study conformed to the Code of Ethics of
the World Medical Association (Declaration of Helsinki)
and was approved by the Ethics Committee of the capital
region of Copenhagen (Region Hovedstaden).
Design
The study lasted 40 days and consisted of 10 sessions of
supervised speed endurance training (SET) and 10 ses-
sions
of
aerobic
moderate-intensity
(AM)
training
(Fig. 1). Total running distance during the intervention
period was reduced (P < 0.05) by 36% compared to
before
the
intervention
(mean  SE,
16  1
vs.
25  2 km/week).
Screening and familiarization
Before being included in the study, subjects performed a
10-km running test and an incremental treadmill test to
exhaustion with pulmonary VO2 measured by a breath-
by-breath gas analyzing system (Oxycon Pro; Viasys
Healthcare, Hoechberg, Germany), and heart rate (Polar
Team2 transmitter; Polar Electro Oy, Kempele, Finland)
collected throughout the test.
Training
SET was performed on day two and six of an 8-day cycle
at Østerbro Stadium, Copenhagen, on an outdoor 400-m
running track. In ﬁrst and ﬁnal SET session, subjects
completed six bouts of 30-sec running. The ﬁrst bout was
performed
with
near-maximal
intensity,
whereas
the
remaining ﬁve bouts were performed with maximal inten-
sity and distance covered was measured. For the remain-
ing eight SET sessions, subjects completed ten bouts of
30-sec “all-out” running. In all sessions, running bouts
were separated by 3.5 min of recovery (walking ~200 m
to the start-line). SET sessions were supervised, but the
subjects performed the SET sessions on their own, if they
were unable to participate in the supervised training
(85  4% adherence to the supervised SET).
AM training was performed on the ﬁrst and ﬁfth day
during the 8-day cycle. These sessions were not super-
vised, but subjects kept a training log to record exercise
distance, time and intensity. A Polar FT7 (Polar Electro
Oy, Kempele, Finland) or personal watch with HR moni-
tor was used to record exercise intensity and training logs
was continuously analyzed. The adherence to the AM
training sessions was 93  3% with a weekly duration of
68  5 min and with an average heart rate of 83  1%
of HRmax.
Testing
Tests were performed on separate days interspersed by at
least 48 hours, on the same treadmill in the Exercise
Physiology laboratory at August Krogh Institute, Depart-
ment of Nutrition, Exercise and Sports, University of
Copenhagen, before and after the intervention. Tests
included: (1) an incremental running test to exhaustion
(INC); (2) repeated bouts of 6-min submaximal running
followed by a 10-km running test on a running track in a
normal condition; (3) repeated bouts of 6-min submaxi-
mal running followed by a 10-km running test on a run-
ning track in a ST glycogen-depleted condition; (4) a
muscle biopsy and a blood sample collected at rest after
an overnight fast (Fig. 1).
All tests were carried out at the same time of day. Sub-
jects refrained from strenuous physical activity, alcohol
and caffeine 24 h before testing. Subjects were instructed
to keep a diary journal 2 days before and during the ﬁrst
series of tests, and to replicate this diet when tested again.
0
8
16
24
32
40
days
Pre testing:
•
INC
•
10-km normal
•
•
10-km depleted
muscle and blood
sampling
•
muscle and blood
sampling
Post testing:
•
INC
•
10-km normal
•
10-km depleted
Figure 1. Testing before (Pre) and after (Post) 5 blocks/40 days of speed endurance training and reduced training volume in trained runners.
Small grey, black and white boxes on the timeline are days with aerobic moderate-intensity training, speed endurance training and rest days,
respectively. INC: incremental test to exhaustion.
ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
2018 | Vol. 6 | Iss. 3 | e13601
Page 3
C. Skovgaard et al.
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
The incremental running test to exhaustion
INC consisted of 2 min of walking at 5 km/h, 6 min at
the subject’s individual average 10-km running pace
determined at the 10-km screening test before the inter-
vention (v10 km; 13.7  0.3 km/h), and 2 min at 14 or
15 km/h (dependent on v10 km), after which the speed
increased by 1 km/h every minute until exhaustion. Dur-
ing INC, VO2-max, deﬁned as the highest average value
achieved over a 30-sec period (Howley et al. 1995), and
maximal
incremental
speed
(vVO2-max)
{[vVO2-
max = Vf + (Ti/60)],
where
Vf
is
the
ﬁnal
velocity
obtained and Ti is the time spent at the ﬁnal speed level}
were determined. Attaining of maximal heart rate (HR)
(judged against the screening test) and an RER value of
>1.15 were used as criterions. During the last part of the
test, the subjects were verbally encouraged to continue
their effort until voluntary termination of the test. Before
the test, body mass was measured and subjects wore a
Polar Team2 HR monitor around their chest for continu-
ous HR recordings. Pulmonary VO2 was measured by use
of Oxycon Pro, which was calibrated prior to each test.
Muscle and blood sampling
Sampling of muscle and blood was performed between 7
and 11 AM after an overnight fast. Using the Bergstr€om
procedure (Bergstrom 1962), a muscle biopsy was col-
lected with a 5-mm needle from a standardized depth of
5 cm in the middle of m. vastus lateralis of the right leg
at rest using local anesthesia (1 mL; 20 mg/L lidocaine
without adrenaline). The muscle sample (~100 mg wet
weight) was immediately frozen in liquid N2 and stored
at 80°C until further analysis. Next, a catheter was
inserted in the antecubital vein, and a ~7-mL blood sam-
ple was collected and stored on ice until being analyzed.
10-km running tests
Both before and after the intervention, two 10-km run-
ning tests were performed on a 400-m outdoor running
track (Østerbro Stadium, Copenhagen) under similar
weather conditions (~20°C, partly cloudy, light winds)
between the beginning of July and end of August. The
10-km tests were conducted in a randomized order either
without (normal) or after a muscle ST glycogen depletion
protocol that was performed the day before the test (see
later). Each 10-km running test was preceded by two
bouts of 6 min of running, separated by 20 min of rest,
on a treadmill at the subject’s individual 60% vVO2-max
(11.6  0.2 km/h) with respiratory and HR measure-
ments. After these bouts, subjects biked to Østerbro Sta-
dium (1-km, slow pace) for the 10-km test.
Muscle slow-twitch glycogen depletion protocol
The protocol was based on the ﬁndings from the study
by Krustrup et al. (2004) who used a 3-h cycling protocol
(~50% VO2-max) to deplete ST ﬁbers the day before 20-
min of submaximal cycling. The authors reported that the
glycogen depletion of ST ﬁbers (51 and 44% of the ST
ﬁbers were empty and almost empty of glycogen, respec-
tively, and less than 2% of the FT ﬁbers were empty of
glycogen) enhanced the recruitment of FT ﬁbers (Krus-
trup et al. 2004). The protocol is veriﬁed by previous
ﬁndings that ST ﬁbers are exclusively active at 50% VO2-
max when subjects have normal muscle glycogen levels
(Gollnick et al. 1974; Vøllestad and Blom 1985).
The subjects completed a 3-h exercise protocol consist-
ing of 60 min of cross-training, 30 min of cycling,
30 min of running, and 60 min of striding at a low speed
to deplete glycogen in ST muscle ﬁbers of the calves and
thigh muscles. The protocol was chosen to minimize
muscle soreness from eccentric contractions while mim-
icking the movement pattern of running. During the pro-
tocol,
subjects’
HR
was
monitored
to
ensure
they
exercised at 60–65% of HRmax (~50% VO2-max). Average
HR during the 3-h depletion protocol was the same
before
and
after
the
intervention
(120  1
vs.
120  1 bpm; 63  0 vs. 63  0% HRmax). The protocol
started at 6:30 PM and ﬁnished around 10:00 PM and sub-
jects were allowed water ad libitum. After termination of
the protocol, subjects were given a diet consisting of 5E%
carbohydrate, 35E% protein, and 60E% fat with a total
energy intake of 30 kJ/kg body mass at dinner and 10 kJ/
kg at breakfast. Breakfast was consumed 2 h before the
10-km running test, which started at 8:00 AM.
Whole muscle protein expression
Western blotting was performed to determine protein
expression
as
described
previously
(Skovgaard
et al.
2014). In short, ~2.5 mg dry weight (dw; freeze-dried for
a minimum of 24 h) of each muscle sample was dissected
free from blood, fat, and connective tissue. Samples were
homogenized for 1 min at 28.5 Hz (Qiagen Tissuelyser II;
Retsch) in a fresh batch of ice-cold buffer containing (in
mM) 10% glycerol, 20 Na-pyrophosphate, 150 NaCl, 50
HEPES (pH 7.5), 1% NP-40, 20 b-glycerophosphate, 2
Na3VO4, 10 NaF, 2 PMSF, 1 EDTA (pH 8), 1 EGTA (pH
8), 10 lg/mL aprotinin, 10 lg/mL leupeptin, and 3 ben-
zamidine, after which they rotated for 1 h at 4°C, and
centrifuged at 18,320g for 20 min at 4°C to exclude
nondissolved structures. The supernatant (lysate) was col-
lected and used for further analysis. Total protein concen-
tration in each sample was determined by a BSA standard
kit (Thermo Scientiﬁc), and samples were mixed with 69
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ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
C. Skovgaard et al.
Laemmli buffer (7 mL 0.5 mol/L Tris-base, 3 mL glycerol,
0.93 g DTT, 1 g SDS, and 1.2 mg bromophenol blue)
and ddH2O to reach equal protein concentration before
protein expression was determined by western blotting.
Equal amounts of total protein (6–12 lg depending on
the protein of interest) were loaded in each well of precast
gels (Millipore). All samples from each subject were loaded
on the same gel. Proteins were separated according to their
molecular weight by SDS-PAGE and semi-dry transferred
to a 0.45 lm PVDF membrane (Bio-Rad). The membranes
were blocked in either 2% skimmed milk or 3% BSA in
TBST, including 0.1% Tween-20 before an overnight incu-
bation with rocking in primary antibody at 4°C. The pri-
mary antibodies used were: (ab. cat number and company,
respectively):
sarcoplasmic
reticulum
Ca2+-ATPase
1
(SERCA1; MA3-912; Thermo Scientiﬁc), sarcoplasmic
reticulum Ca2+-ATPase 2 (SERCA2; N-19 Sc-8095; Santa
Cruz Technology), actin (A2066; Sigma Aldrich), mito-
chondrial uncoupling protein 3 (UCP3; AB3046; Milli-
pore). The membranes were then incubated for 1 h at
room temperature in horseradish peroxidase conjugated
secondary antibody (rabbit anti-sheep (P-0163, DAKO),
rabbit anti-goat (P-0449, DAKO), goat anti-mouse (P-
0447, DAKO) or goat anti-rabbit IgM/IgG (4010-05; South-
ern Biotech), depending on the primary antibody source).
The protein bands were visualized with ECL (Millipore)
and recorded with a digital camera (ChemiDoc MP Imag-
ing System, Bio-Rad Laboratories). For each muscle sam-
ple, protein expression was determined in duplicate on
individual gels. Quantiﬁcation of the band intensity was
performed using Image Lab version 4.0 (Bio-Rad Labora-
tories). Each band was normalized to two control samples
of human, whole-muscle homogenate that were loaded
onto every gel.
Single muscle ﬁber protein expression
To determine the protein expression of citrate synthase
(CS), UCP3 as well as SERCA- and myosin heavy chain
(MHC) isoforms in different muscle ﬁber types, 88  5
single-ﬁber segments were collected from each freeze-dried
muscle biopsy. Individual segments were isolated under a
microscope at room temperature using ﬁne jeweler’s for-
ceps, and were individually incubated for 1 h at room tem-
perature
in
microfuge
tubes
containing
10 lL
of
denaturing buffer (0.125 mol/L Tris-HCl, 10% glycerol,
4% SDS, 4 mol/L urea, 10% mercaptoethanol, and 0.001%
bromophenol blue, pH 6.8) (Murphy, 2011). The dena-
tured segments were stored at 80°C until being analyzed
for ﬁber type and grouped accordingly as described below.
The ﬁber type of ﬁber segments was determined using
dot blotting. 1.5 lL of each denatured sample was spotted
onto two PVDF membranes, which were pre-activated in
95%
ethanol
and
pre-equilibrated
in
transfer
buffer
(25 mmol/L Tris, 192 mmol/L glycine, pH 8.3, 20%
methanol). After drying completely at room temperature,
the membranes containing samples were reactivated in
ethanol and re-equilibrated in transfer buffer, before
being blocked in 5% skim milk in TBST for 5–30 min.
One membrane was then incubated by gentle rocking
with MHCI antibody (1:200 in 1% BSA with PBST;
mouse monoclonal IgM, clone A4.840, Developmental
Studies Hybridoma Bank (DSHB)), and the other with
MHCIIa antibody (mouse monoclonal IgG, clone A4.74,
DSHB) for 2 h at room temperature. After a quick wash
in TBST, secondary antibody was applied (1:10,000), and
protein signals quantiﬁed as described under Whole mus-
cle protein expression (section above).
The remaining part of each denatured ﬁber segment
(7 lL) was pooled into groups of ST or FTa ﬁbers depend-
ing on MHC expression. The number of segments entailed
in each pool of ﬁbers per biopsy was 15  2 (range: 8–42)
for ST and 18  2 (range 8–39) for FTa ﬁbers before the
intervention, and 19  3 (range: 7–55) and 18  2 (range
7–41), respectively, after the intervention. Hybrid ﬁbers
(expressing multiple MHC isoforms) were excluded from
analysis. Protein expression was determined in pools of ST
and FTa ﬁbers using western blotting as detailed in the sec-
tion above. The primary antibodies used were: (ab. cat
number and company, respectively): CS (ab96600, Abcam),
UCP3 (AB3046; Millipore) SERCA1 (MA3-912; Thermo
Scientiﬁc), SERCA2 (N-19 Sc-8095; Santa Cruz Technol-
ogy). Pools of ﬁbers from biopsies obtained before and
after the intervention was loaded on the same gel (stain-
free, 4–15%, precast), along with either a calibration curve
or two loading controls of whole-muscle homogenate. Pro-
tein bands were quantiﬁed by normalizing each band to the
total protein content in each lane on the stain-free gel.
Muscle enzyme activity
Muscle
enzyme
activity
was
determined
by
use
of
~2.5 mg dw muscle tissue dissected free from blood, fat,
and connective tissue, which was homogenized (1:400) in
a 0.3 mol/L phosphate buffer (pH 7.7) by 2 rounds of
30-sec using a TissueLyser II (Retch, Germany). Maximal
activity of CS, b-hydroxyacyl-CoA-dehydrogenase (HAD)
and phosphofructokinase (PFK) was determined ﬂuoro-
metrically with NAD-NADH coupled reactions (Lowry
and Passonneau 1972) on a Fluoroskan Ascent apparatus
(Thermo Scientiﬁc) using Ascent Software version 2.6.
Blood analysis
A total of ~7 mL blood was drawn in a heparinized 2-mL
syringe and a 5-mL syringe at rest. A part of the 2-mL blood
ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
2018 | Vol. 6 | Iss. 3 | e13601
Page 5
C. Skovgaard et al.
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
sample (~1.5 mL) and the 5-mL sample (split into
2 9 2 mL tubes containing 30 lL EDTA) were centrifuged
at 20,000 g for ~2 min and the remaining whole blood from
the 2-mL sample (~0.5 mL) was stored on ice for further
analyses. After centrifugation, the plasma was transferred
into tubes that were placed in ice-cold water until they were
stored at 20°C. Plasma samples were subsequently ana-
lyzed for testosterone and cortisol, creatine kinase (CK) and
immunoglobulin A (IgA). CK activity was analyzed by enzy-
matic kinetic assay methods (Roche Diagnostic, Mannheim,
Germany) using a Hitachi 912 (Roche Diagnostic, Indi-
anapolis). IgA was determined using an immunoturbidi-
metric assay method (Horiba, Montpellier, France) on an
automatic analyzer (Pentra C400, Horiba, Montpellier,
France). Testosterone and cortisol was determined using
ELISA kits (R&D Systems, Inc. Minneapolis). Whole blood
was analyzed for hemoglobin, hematocrit and HCO3 at rest
(ABL800 Flex; Radiometer Medical, Copenhagen, Den-
mark).
Running economy
Running Economy (RE) was calculated using the follow-
ing formula:
REðmLO2=kg=kmÞ ¼ VO2ðmL/minÞ  60 min/h=BM (kg)
 running speedðkm/hÞ
where VO2 is the average value during the last 2 min of
running for the two intervals at 60% vVO2-max and v10-
km, and BM is body mass.
Statistics
Paired t tests were used to evaluate the effect of the inter-
vention (Pre vs. Post) with two-way ANOVA repeated mea-
sures being used to evaluate the effect of glycogen condition
(normal vs. ST glycogen-depleted) on 10-km running per-
formance and running economy (at 60% vVO2-max). Level
of signiﬁcance was set at P < 0.05. A Student-Newman
Keuls post-hoc test was applied in case signiﬁcance was
reached in the ANOVA. Absolute data values was used and
presented as means  SE unless otherwise stated.
Results
Pulmonary oxygen uptake and heart rate
during submaximal exercise
Pulmonary VO2 during running at v10-km was 1.9%
lower (P < 0.05) after compared to before the interven-
tion (3.46  0.14 vs. 3.53  0.14 L/min), and running
economy was improved by 2.1% (P < 0.05; 207.6  2.6
vs. 212.1  2.8 mL/kg/km) (Fig. 2). Mean HR at v10-km
was 1.7% lower (P < 0.05) after than before the interven-
tion (162  2 vs. 165  2 bpm).
In the normal condition, pulmonary VO2 at 60%
vVO2-max was the same before and after the intervention
(3.01  0.13 vs. 2.99  0.13 L/min), whereas running
economy was 1.7% better (P < 0.05) after compared to
before the intervention (210.4  2.9 vs. 214.1  3.2 mL/
kg/km) (Fig. 3). In the ST glycogen-depleted condition,
pulmonary VO2 at 60% vVO2-max (3.05  0.15 (Post)
vs. 3.04  0.13 (Pre) L/min) and running economy
(216.5  2.9 (Post) vs. 217.4  2.9 (Pre) mL/kg/km) did
not change with the intervention (Fig. 3).
Before the intervention, pulmonary VO2 at 60% vVO2-
max was the same in normal and ST glycogen-depleted
condition, whereas after the intervention, pulmonary VO2
was 2.0% lower (P < 0.01) in normal than ST glycogen-
depleted condition. Before and after the intervention,
running economy was 1.6% and 2.9% better (P < 0.05),
respectively, in the normal compared to the ST glycogen-
depleted condition (Fig. 3).
HR during running at 60% vVO2-max in normal and
ST glycogen-depleted condition did not change with the
intervention, and there were no differences between
conditions.
Expression of proteins in muscle
homogenate
Expression of SERCA2 in muscle homogenate was 20%
higher (P < 0.05) after compared to before the interven-
tion, whereas expression of muscle SERCA1 was 22%
lower (P < 0.05). Expression of muscle actin and UCP3
did not change with the intervention (Fig. 4).
Expression of proteins in single muscle
ﬁbers
After compared to before the intervention, expression of
muscle CS and UCP3 in ST ﬁbers was 22% and 25%,
respectively, lower (P < 0.05), and expression of muscle
dystrophin in ST ﬁbers was 41% higher (P < 0.05)
(Fig. 5). Expression of muscle SERCA1 was 19% lower
(P < 0.05) in FTa ﬁbers, and expression of MHCIIa was
19% higher (P < 0.05) in FTa ﬁbers after than before the
intervention. Expression of SERCA2 and MHCI in the
single ﬁber pools was unchanged with the intervention
(Fig. 5).
Muscle enzymatic activity
Maximal activity of CS, HAD, and PFK was 10.7%, 9.1%,
and 23.4%, respectively, higher (P < 0.05) after than
before the intervention (Table 1).
2018 | Vol. 6 | Iss. 3 | e13601
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ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
C. Skovgaard et al.
10-km run
Compared to before, 10-km performance in the normal
condition improved (P < 0.01) by 3.2% (43.7  1.0 vs.
45.2  1.2 min) after the intervention (Fig. 6). In the ST
glycogen-depleted
condition,
10-km
performance
was
3.9% better (P < 0.001) after compared to before the
intervention (45.8  1.2 vs. 47.7  1.3 min; Fig. 6). Ten
kilometer performance was reduced (P < 0.001) to the
same degree in the ST glycogen-depleted compared to
the normal condition before (5.3%) and after (4.7%) the
intervention (Fig. 6).
Maximum oxygen uptake, body mass, and
heart rate
VO2-max was the same before and after the intervention
(4.06  0.16
vs.
4.13  0.18 L/min;
56.4  1.0
vs.
56.3  1.2 mL/min/kg), but vVO2-max was 2.0% higher
(P < 0.05) after compared to before (19.3  0.3 vs.
18.9  0.3 km/h). Peak heart rate during INC was the
same before and after the intervention (187  2 vs.
188  2 bpm) as well as body mass (72.5  2.4 vs.
72.9  2.3).
Blood variables
Blood hematocrit and concentration of hemoglobin as
well as plasma concentrations of testosterone, cortisol, CK
and HCO3
 were the same before and after the interven-
tion. Compared to before the intervention, testosterone to
cortisol ratio was 31.3% higher (P < 0.05) and plasma
IgA level was 4.0% higher (P < 0.05) after (Table 2).
Discussion
The main ﬁndings of the present study were that a period
of intense and reduced volume of training in trained
202
204
206
208
210
212
214
216
Pre
Post
Running economy (ml/kg/km) at v10-km
B
***
3350
3400
3450
3500
3550
3600
3650
3700
Pre
Post
Oxygen uptake (L/min) at v10-km
A
***
Figure 2. Oxygen uptake (A) and running economy (B) at v10-km before (Pre) and after (Post) 5 blocks/40 days of speed endurance training
and reduced training volume in trained runners. Values are means  SE. ***Post different (P < 0.001) to Pre.
204
206
208
210
212
214
216
218
220
222
Pre
Post
Running Economy (ml/kg/km) 
at 60% VO2-max
Depleted
Normal
B
#
#
*
2850
2900
2950
3000
3050
3100
3150
3200
3250
Pre
Post
Oxygen uptake (L/min) 
at 60% VO2-max
Depleted
Normal
A
#
Figure 3. Oxygen uptake (A) and running economy (B) at 60% VO2-max in depleted and normal conditions before (Pre) and after (Post) 5
blocks/40 days of speed endurance training and reduced training volume in trained runners. Values are means  SE. *Post different (P < 0.05)
to Pre; #difference (P < 0.05) within time-point.
ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
2018 | Vol. 6 | Iss. 3 | e13601
Page 7
C. Skovgaard et al.
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
runners improved running economy together with higher
expression of dystrophin and lowered expression of UCP3
in ST muscle ﬁbers as well as lower expression of
SERCA1 in FTa muscle ﬁbers. In addition, compared to
the normal condition, 10-km running performance and
running economy was equally reduced after the ST mus-
cle glycogen-depletion protocol before and after the inter-
vention period.
The better running economy at 60% vVO2-max and
v10-km after the intervention period is in accordance
with ﬁndings in other studies of intense training and low-
ered training volume in trained runners (Bangsbo et al.
2009; Iaia and Bangsbo 2010; Skovgaard et al. 2014). In
the
ST glycogen-depleted
condition,
where
a higher
recruitment of FT ﬁbers would be expected, the running
economy remained unchanged with training, suggesting
that it was mainly changes in ST ﬁbers that caused the
improvement in running economy in the normal condi-
tion. In accordance, the expression of UCP3 in ST ﬁbers
was lowered by training in the present study. As mechani-
cal energy efﬁciency is negatively related to UCP3 expres-
sion (Russell et al. 2003a,b; Mogensen et al. 2006), this
suggests that the reduced UCP3 expression in ST ﬁbers
may have improved the mitochondrial efﬁciency, and
thereby running economy. On the other hand, reduced
energy
expenditure
during
submaximal
exercise
was
–50%
–40%
–30%
–20%
–10%
0%
10%
20%
30%
40%
50%
60%
Actin
UCP3
SERCA1
SERCA2
Muscle expression (Post relative to Pre)
*
*
Figure 4. Protein expression of actin; UCP3, mitochondrial
uncoupling protein 3; SERCA1 and 2, sarcoplasmic reticulum
calcium ATPase, before (Pre) and after (Post) 5 blocks/40 days of
speed endurance training and reduced training volume in trained
runners. Values are geometric means  95% conﬁdence interval
(CI) (Post relative to Pre). *Post different (P < 0.05) from Pre.
40%
60%
80%
100%
120%
140%
160%
180%
MHCIIa
SERCA1
CS
UCP3
Dystrophin
FTa single muscle fiber expression (relative to Pre)
Post FTa
*
*
40%
60%
80%
100%
120%
140%
160%
180%
MHCI
SERCA2
CS
UCP3
Dystrophin
ST single muscle fiber expression (relative to Pre)
Post ST
*
*
*
A
B
Figure 5. ST (A) and FTa (B) single muscle ﬁber expression of MHCI and II, myosin heavy chain; SERCA1 and 2, sarcoplasmic reticulum calcium
ATPase; CS, citrate synthase; UCP3, mitochondrial uncoupling protein 3; and dystrophin, before (Pre) and after (Post) 5 blocks/40 days of speed
endurance training and reduced training volume in trained runners. Values are means  SE (relative to Pre). *Post different (P < 0.05) from Pre.
Table 1. Maximal
activity
of
muscle
citrate
synthase
(CS),
b-hydroxyacyl-CoA-dehydrogenase
(HAD);
phosphofructokinase
(PFK) at rest before (Pre) and after (Post) 5 blocks/40 days of
speed endurance training and reduced training volume in trained
runners.
Pre
Post
CS (lmolg/dw/min)
17.7  2.9
19.6  2.9*
HAD (lmolg/dw/min)
15.6  0.9
17.0  0.7*
PFK (lmolg/dw/min)
72.1  15.3
88.9  13.7*
Data are presented as means  SE.
*Post different (P < 0.05) to Pre.
2018 | Vol. 6 | Iss. 3 | e13601
Page 8
ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
C. Skovgaard et al.
reported in a study where trained subjects (VO2-max:
56  1 mL/min/kg) performed 4 weeks of speed endur-
ance training (8–12 9 30-sec at maximum
speed; 3
times/week) with a 65% reduced training volume without
change in the expression of whole muscle UCP3 (Iaia
et al. 2009). It could be speculated that the reduction in
training volume was too large to elicit changes in UCP3
expression or that a potential reduced expression of
UCP3 in ST ﬁbers was undetected by the analysis of
whole muscle tissue (Iaia et al. 2009).
In addition, the expression of dystrophin, a protein
that connects the sarcomere and the extracellular matrix
(Hughes et al. 2015), increased in ST muscle ﬁbers with
the training intervention. An important function of dys-
trophin is to transmit forces generated by the actin-myo-
sin cross-bridge (Chopard et al. 2005), and the higher
expression of dystrophin in ST ﬁbers may have enhanced
the structural integrity of the ST muscle ﬁbers and
thereby inﬂuenced running economy.
The intervention period also led to lowered expression
of SERCA1 in FT muscle ﬁbers, which has also been
found in studies of endurance training (Majerczak et al.
2008, 2012; Green et al. 2011). The lower expression of
muscle SERCA1 may have reduced the energy turnover
during exercise, since calcium handling by the ATP
dependent SERCA pumps is reported to be responsible
for up to 50% of total energy usage (Clausen et al. 1991;
Walsh et al. 2006; Smith et al. 2013), and, thus, may have
contributed to the better running economy after the
intervention period.
The ﬁnding of improved 10-km running performance
after the intervention period is in agreement with other
studies investigating the effect of intense training and low-
ered training volume in trained runners (Bangsbo et al.
2009; Iaia and Bangsbo 2010; Skovgaard et al. 2014). The
novel ﬁnding in the present study was that the magnitude of
the difference between 10-km running in normal versus ST
ﬁber glycogen-depleted condition was the same before and
after the training period. This observation suggests that any
effect of the intervention on the oxidative capacity of the FT
ﬁbers was small, which is supported by the ﬁnding that the
expression of CS in the FT ﬁbers did not change with the
intervention. In agreement, a 7-week intense training period
(12 9 30-sec maximal sprints 2.5 times/week and 5 9 4-
min intervals (at a heart rate (HR) of 89% HRmax) 1.5
times/week) with a 50% reduction in training volume, did
not change expression of muscle CS and COX-4 in segments
of FT ﬁbers in well trained cyclists (VO2-max: 59  4 mL/
min/kg) (Christensen et al. 2015). Collectively, these ﬁnd-
ings suggest that intense training with a decrease (36–50%)
in training volume does not affect oxidative proteins in FT
muscle ﬁbers in trained subjects. Nevertheless, the mixed
muscle CS activity was elevated with the intervention and
may have contributed to the better 10-km performance.
In agreement with other studies on the effect of speed
endurance training and reduced training volume in run-
ners (Bickham et al. 2006; Iaia et al. 2008; Bangsbo et al.
2009; Iaia and Bangsbo 2010; Skovgaard et al. 2014),
VO2-max did not change with the intervention and can-
not explain the improved 10-km performance. Based on
the performance during the 10-km run, VO2-max and
running economy, the fraction of FVO2-max [FVO2-
max = 10-km velocity (km/hr)*running economy at v10-
km (mL/kg/km)/VO2-max (mL/min/kg)100] during the
10-km run was calculated. It showed that FVO2-max did
not
change
with
the
intervention
period
(Pre:
84.1  1.3% vs. Post: 85.1  1.2%). In agreement, Iaia
et al. (2009) observed a FVO2-max of 84.8% and 81.6%
at v10-km (14.5 km/h) before and after, respectively, a 4-
wk intervention period with speed endurance training.
41
42
43
44
45
46
47
48
49
50
Pre
Post
10-km time (min)
Depleted
Normal
**
**
###
###
Figure 6. Time to complete a 10-km run in depleted and normal
conditions before (Pre) and after (Post) 5 blocks/40 days of speed
endurance training and reduced training volume in trained runners.
Values are means  SE. **Post different (P < 0.01) to Pre;
###difference (P < 0.001) within time-point.
Table 2. Plasma testosterone (T), cortisol (C) and T:C ratio, hemo-
globin and hematocrit, creatine kinase, (CK), and Immunoglobulin
A, (IgA) and HCO3
 before (Pre) and after (Post) 5 blocks/40 days
of speed endurance training and reduced training volume in
trained runners.
Pre
Post
Testosterone (nmol/L)
24.2  2.9
25.4  3.5
Cortisol (nmol/L)
195.8  21.2
168.3  25.2
t:c ratio
0.16  0.03
0.21  0.03*
Hemoglobin (mmol/L)
8.9  0.2
8.5  0.2
Hematocrit (%)
44.2  1.1
41.8  0.9
CK (U/L)
208  41
155  17
IgA (g/L)
1.98  0.19
2.06  0.19*
HCO3
 (mmol/L)
24.3  0.4
25.0  0.7
Data are presented as means  SE.
*Post different (P < 0.05) to Pre.
ª 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society
2018 | Vol. 6 | Iss. 3 | e13601
Page 9
C. Skovgaard et al.
Muscle Fiber Type Adaptations to Speed Endurance Training in Runners
And in the study by Bangsbo et al. (2009), FVO2-max
was 85.7% and 83.6% at v10-km (16.0 km/h) before and
after, respectively, a 6–9-week period with speed endur-
ance training and a basic volume of aerobic training in
trained runners. These observations suggest that changes
in FVO2-max do not explain the improved 10-km perfor-
mance with speed endurance training and reduced train-
ing volume. Thus, the improved performance of the 10-
km run appears mainly to be caused by the better run-
ning economy. It should be noted, however, that the
anaerobic energy production during the 10-km run,
which is suggested to amount up to 20% of the energy
provided during a 10-km run (Joyner and Coyle 2008), is
not taken into account in the calculation. In the present
study, anaerobic energy production may have been higher
after the speed endurance training period due to a possi-
ble higher anaerobic capacity reﬂected by the ﬁnding of
unchanged VO2-max and higher maximal speed during
the incremental test. In support, maximal activity of PFK
was higher after the intervention period, which theoreti-
cally may have promoted a higher energy production
from glycolysis during the 10-km run.
In summary, running economy was improved after
40 days of intense and reduced volume of training, which
may have been related to a reduced expression of UCP3
and higher expression of dystrophin in ST muscle ﬁbers
and lower expression of SERCA1 in FT muscle ﬁbers. The
ﬁnding that running economy at 60% VO2-max in a ST
muscle ﬁber glycogen-depleted condition was unchanged,
suggests that the change in running economy was due to
adaptation in ST muscle ﬁbers. The better running econ-
omy may explain the improved 10-km running perfor-
mance together with a possibly higher anaerobic capacity.
Acknowledgments
We thank all the participants for their valuable time and
extraordinary effort during training and testing. We also
gratefully acknowledge Jens Jung Nielsen and Jon Egelund
for excellent technical assistance and Thomas Gunnarsson,
Morten Hostrup, Lars Nybo, Johan Onslev, Sajad Habib,
Steffen Raun, Julian Christoffer Bachmann, Peter Munch
Larsen and Stefan Madsen for invaluable help during
training and experimental days.
Conﬂict of Interest
None declared.
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C. Skovgaard et al.

Effect of speed endurance training and reduced training volume on running economy and single muscle fiber adaptations in trained runners.
Skovgaard, Casper,Christiansen, Danny,Christensen, Peter M,Almquist, Nicki W,Thomassen, Martin,Bangsbo, Jens