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Skeletal muscle adaptation: Training twice every second day vs. training once daily


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Low muscle glycogen content has been demonstrated to enhance transcription of a number of genes involved in training adaptation. These results made us speculate that training at a low muscle glycogen content would enhance training adaptation. We therefore performed a study in which seven healthy untrained men performed knee extensor exercise with one leg trained in a low-glycogen (Low) protocol and the other leg trained at a high-glycogen (High) protocol. Both legs were trained equally regarding workload and training amount. On day 1, both legs (Low and High) were trained for 1 h followed by 2 h of rest at a fasting state, after which one leg (Low) was trained for an additional 1 h. On day 2, only one leg (High) trained for 1 h. Days 1 and 2 were repeated for 10 wk. As an effect of training, the increase in maximal workload was identical for the two legs. However, time until exhaustion at 90% was markedly more increased in the Low leg compared with the High leg. Resting muscle glycogen and the activity of the mitochondrial enzyme 3-hydroxyacyl-CoA dehydrogenase increased with training, but only significantly so in Low, whereas citrate synthase activity increased in both Low and High. There was a more pronounced increase in citrate synthase activity when Low was compared with High. In conclusion, the present study suggests that training twice every second day may be superior to daily training.
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doi: 10.1152/japplphysiol.00163.2004
98:93-99, 2005. First published 10 September 2004;J Appl Physiol
Saltin and Bente Klarlund Pedersen
Anne K. Hansen, Christian P. Fischer, Peter Plomgaard, Jesper Løvind Andersen, Bengt
day vs. training once daily
Skeletal muscle adaptation: training twice every second
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Skeletal muscle adaptation: training twice every second day
vs. training once daily
Anne K. Hansen, Christian P. Fischer, Peter Plomgaard,
Jesper Løvind Andersen, Bengt Saltin, and Bente Klarlund Pedersen
Department of Infectious Diseases and The Copenhagen Muscle Research Centre,
Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Submitted 13 February 2004; accepted in final form 7 September 2004
Hansen, Anne K., Christian P. Fischer, Peter Plomgaard, Jes-
per Løvind Andersen, Bengt Saltin, and Bente Klarlund Ped-
ersen. Skeletal muscle adaptation: training twice every second day vs.
training once daily. J Appl Physiol 98: 93–99, 2005. First published
September 10, 2004; doi:10.1152/japplphysiol.00163.2004.—Low
muscle glycogen content has been demonstrated to enhance transcrip-
tion of a number of genes involved in training adaptation. These
results made us speculate that training at a low muscle glycogen
content would enhance training adaptation. We therefore performed a
study in which seven healthy untrained men performed knee extensor
exercise with one leg trained in a low-glycogen (Low) protocol and
the other leg trained at a high-glycogen (High) protocol. Both legs
were trained equally regarding workload and training amount. On day
1, both legs (Low and High) were trained for 1 h followed by2hof
rest at a fasting state, after which one leg (Low) was trained for an
additional 1 h. On day 2, only one leg (High) trained for 1 h. Days 1
and 2 were repeated for 10 wk. As an effect of training, the increase
in maximal workload was identical for the two legs. However, time
until exhaustion at 90% was markedly more increased in the Low leg
compared with the High leg. Resting muscle glycogen and the activity
of the mitochondrial enzyme 3-hydroxyacyl-CoA dehydrogenase in-
creased with training, but only significantly so in Low, whereas citrate
synthase activity increased in both Low and High. There was a more
pronounced increase in citrate synthase activity when Low was
compared with High. In conclusion, the present study suggests that
training twice every second day may be superior to daily training.
substrate availablity; 3-hydroxyacyl-CoA dehydrogenase; citrate syn-
area within exercise physiology for many years. When relating
to the effect on performance, e.g., running time in a marathon
race, it is clear that carbohydrate loading the days before to
rebuild muscle glycogen as well as carbohydrate intake during
the race enhance performance (28, 31, 37). In analogy, a
common belief is that carbohydrate intake during training at
high amounts will allow the athlete to train harder and longer
and thus achieve a superior training response. However, this
argument does not consider the unsolved longstanding question
of whether it is a lack or a surplus of a substrate that triggers
the training adaptation (14).
In endurance exercise, adaptation includes systemic changes
such as improved maximal oxygen uptake (V
2 max
). Adapta
tion also includes prolonged time until exhaustion at a given
workload, which is linked to maximal aerobic power but may
also be linked to local factors within the muscle (46). The
multitude of adaptations that occur with training that allow for
greater performance also includes an increased number of
capillaries (2, 3), an increased density of mitochondria with the
activity of enzymes such as 3-hydroxyacyl-CoA dehydroge-
nase (HAD) and citrate synthase (CS) being elevated, an
increased concentration of transport proteins, greater glycogen
concentration (24, 27, 30, 46), and a relative increase in the
occurrence of type IIA fibers at the expense of type IIX fibers
(2, 3). As a result, the ability to metabolize fat is enhanced (24).
When a more molecular view on training adaptation is taken,
it is obvious that adaptation is a consequence of accumulation
of specific proteins. The gene expression that allows for these
changes in protein concentration is pivotal to the training
adaptation. Recent studies have demonstrated that exercise
induces transcription of several genes (43). Furthermore, it has
been demonstrated that muscle glycogen is a determining
factor for the transcription of some genes. Exercising when
muscle glycogen concentration was low resulted in a greater
transcriptional activation of interleukin-6 (32), pyruvate dehy-
drogenase kinase 4 (23, 42), hexokinase (42), and heat shock
protein 72 (22) compared with when muscle glycogen concen-
tration was high or normal at the start of exercise. The role of
glycogen could be explained by the fact that several transcrip-
tion factors include glycogen-binding domains. When muscle
glycogen is low, these factors are released and become free to
associate with different targeting proteins (4, 18, 44, 48, 49).
Helge and Kiens (29) studied the role of substrate availabil-
ity on muscle enzyme activity and found that HAD increased
with training after adaptation to a fat-rich diet but not a
carbohydrate-rich diet. Transcriptional activities of HAD and
CS are only markedly influenced by acute muscle contractions
(43). However, it is possible that the accumulation of mRNA
for these genes peaks late in the recovery from the exercise and
that a low muscle glycogen level may enhance the transcription
of these genes.
On this molecular background, we have formulated the
overall hypothesis that training on a low muscle glycogen level
will improve training adaptation (21). In the present study, we
specifically tested the hypothesis that training at a low muscle
glycogen content would enhance levels of HAD and CS.
Moreover, performance defined as time until exhaustion at a
given power output would be more pronounced by training
twice every second day compared with training once daily.
Address for reprint requests and other correspondence: B. K. Pedersen,
Dept. of Infectious Diseases M7641, and The Copenhagen Muscle Research
Centre, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
J Appl Physiol 98: 93–99, 2005.
First published September 10, 2004; doi:10.1152/japplphysiol.00163.2004.
8750-7587/05 $8.00 Copyright
2005 the American Physiological Societyhttp://www. 93
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We therefore designed a protocol of two different training
regimens in which the cycling of muscle glycogen differed.
Critical would be to have direct comparison between legs on
the same individual, where one leg has trained markedly more
with low glycogen than the other. This can be accomplished by
having one leg train twice every second day, whereas the other
only trains once daily.
When a subject exercises, muscle glycogen declines and is
slowly restored over the following 24 h if carbohydrate intake
is normal (14, 26, 34). Therefore, when two exercise sessions
of1hisseparated by 2 h, the second bout of exercise is
undertaken with low muscle glycogen at its start, whereas
muscle glycogen is restored before each exercise bout when the
exercise is separated by 24 h.
Seven healthy untrained young men were recruited with a mean age
of 26 yr (range 24 –29 yr), a mean weight of 86.7 kg (range 53–129
kg), mean height of 180.1 cm (range 171–189 cm). The subjects were
exposed to a highly demanding and intensive training program lasting
10 wk with one- and/or two-legged knee extensor exercise (Fig. 1).
The study was opposed by the local Ethical Committee of Copenha-
gen and Frederiksburg Communities and was performed in accor-
dance with the Declaration of Helsinki. The two legs were trained
after different schedules. By randomization, all subjects trained one
leg twice every second day [low-glycogen training (Low)], whereas
the other leg was trained once daily [high-glycogen training (High)].
Each bout of exercise lasted 1 h. On day 1, both legs trained
simultaneously for1hat75%ofmaximal power output (P
followed by2hofrecovery. Thereafter, the Low leg trained for 1 h
at 75% of P
.On day 2, the High leg trained alone for1hat75%
of P
. This 2-day training cycle was repeated for 10 wk. Every
week, subjects trained 5 days and then rested 2 days. The training
sessions were performed in the morning after an overnight fast, the
first exercise bout being undertaken between 6:00 and 9:00 AM. The
subjects were fasting until the training session was finished. Water
intake was ad libitum. The workload of the 10-wk training was
initially 75% of P
before the training started. Workload was
increased individually by 5–10% depending on the progress of each
individual. The workload was identical for the two legs. Before and
after the 10 wk of training, a P
test was obtained for each leg
separately using the same cycle ergometer-knee extensor exercise
apparatus (Monark, Varberg, Sweden). Thereafter, a 90% P
durance test was performed for each leg.
In the first trial, the subjects performed a V
2 max
test on each leg
to determine the individual knee P
. Both legs were tested on the
same day with at least 30 min of rest in between. The test began with
a 10-min warm-up at 20 W followed by a stepwise increase of 10 W
in workload every 2 min. Subjects worked until exhaustion.
Diet. The subjects consumed a mixed Western diet throughout the
study (16 MJ per day, 70% carbohydrate, 15% protein, 15% fat).
Subjects were asked to adhere to the diet and to refrain from strenuous
exercise other than that included in the training protocol.
Muscle biopsies. Muscle biopsy samples were obtained before and
after 10 wk of training from the vastus lateralis muscle of both legs.
The volunteers were told to abstain from any strenuous exercise 48 h
before these biopsies. In addition, muscle biopsies were obtained in
relation to training sessions (before, immediately after the first bout of
exercise, after2hofrest, and immediately after the second bout of
exercise). Muscle biopsies were analyzed for glycogen by using
enzymatic analyses with fluorometric detection (19). In addition,
biopsies were analyzed for CS and HAD activity (20).
Hormones. Glucose and lactate were measured by use of an
automated analyzer (Cobas Fara, Roche, Basel, Switzerland). Plasma
insulin (Insulin RIA 100, Amersham Pharmacia Biotech, Uppsala,
Sweden), glucagon (Linco Research, St. Charles, MO), and cortisol
(Diagnostic Products, Los Angeles, CA) were determined by RIA, and
plasma epinephrine and norepinephrine were determined by HPLC
(9, 47).
Fiber types and capillaries. Serial sections (10 m) of the muscle
biopsy samples were cut in a cryostat at 20°C, and routine ATPase
histochemistry analysis performed after preincubation at pH 4.37,
4.60, and 10.30 (10). Five different fiber types were defined: types I,
I/IIA, IIA, IIAX, and IIX. The terms “IIAX” and “IIX” have been
used instead of “IIAB” and “IIB,” to match the predominant
nomenclature used for the human myosin heavy chain (MHC)
isoforms (1). Fibers determined to be type II fibers, but showing an
intermediate staining with pH 4.60 preincubation, were categorized
as type IIAX fibers. These fibers covered a wide range from fibers
with only a light staining (i.e., fibers with predominately MHC IIA
expression) to fibers with a much darker staining (i.e., fibers with
predominantly MHC IIX expression). In some individuals, the
number of the minor fiber types (I/IIA, IIAX, and IIX) were so
small that a reliable statistical comparison of changes in fiber-type
size was impossible. Therefore, calculations of fiber-type size were
performed for three major categories of fiber types (type I, type
IIA, and type IIAX). Staining of capillaries was performed by
using the double-staining method (45).
The serial sections of the various ATPase and capillary stainings
were visualized and analyzed for fiber-type percent, fiber-type area
percent, fiber size, and capillary density expressed as capillaries per
fiber and as capillaries per millimeter squared by using a TEMA
image analyzing system (Scanbeam, Hadsund, Denmark) as used
Fig. 1. Schematic overview showing the design of the study. Before onset of the training period, the maximal power output (P
) and the time to exhaustion
) at 90% of P
were determined for each leg on separate occasions. At least 48 h after the last performance test, a blood sample as well as a muscle biopsy
from the vastus laterialis (gray arrows) were obtained during rest after an overnight fast. After the first set of performance tests, the participants trained both legs
for 10 wk, followed by a second posttraining set of performance tests similar to those performed before training. The training consisted of a 14-day cycle repeated
5 times. During one 14-day period (box at top), one leg (Low) was trained for 5 days by the subjects performing 2 bouts of dynamic knee extensor exercise,
each bout lasting 1 h and separated by a 2-h break. Meanwhile, the other leg (High) was trained every day for 5 days/wk by the subjects performing 1 bout of
dynamic knee extensor exercise for 1 h. Numbers in the box at top denote the number of the day during the 14-day training cycle. The workload was initially
set to 75% of the pretraining P
, and increased by 5–10% every 14-days, depending on the progress of each participant. Of note, the workload for each leg
was equal during the training sessions; thus the total work for each leg during the training period was equal.
F, One 1-hour training bout; E, contralateral resting leg.
J Appl Physiol VOL 98 JANUARY 2005
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previously by our laboratory (45). We examined an average of 105
16 fibers in each biopsy.
and time until exhaustion. In response to 10 wk of
training, P
increased significantly, being the same in the two
legs (Table 1). The endurance at 90% of this new P
markedly increased for both legs, but time until exhaustion was
twice as long for Low compared with High. In accordance, the
actual work performed by Low was also markedly larger
compared with High.
Hormones and lactate in relation to a training session.
Plasma insulin, glucagon, norepinephrine, epinephrine, corti-
sol, and lactate were measured before and after1hofknee
extensor exercise with both legs, which corresponded to the
first exercise at that particular day; before and after knee
extensor exercise for 1 h with the Low leg, which was per-
formed 2 h after the exercise with both legs; as well as before
and after1hofexercise with the High leg, which corresponded
to the first bout of exercise on the following day. During both
exercises with two legs as well as one leg, plasma insulin
decreased, whereas plasma glucaogon, norepinephrine, and
epinephrine increased (Table 2). These changes occurred to the
same extent when exercise was performed with two legs and
when exercise was performed with the Low leg. The hormonal
responses to exercise with the Low leg were in general more
pronounced compared with exercise performed with the High
leg. The difference in responses between the Low and High
legs was significant for norepinephrine and epinephrine.
Plasma cortisol did not change in response to exercise.
Muscle glycogen. Muscle glycogen content was measured at
rest before, after 5 wk, and after 10 wk of training (Fig. 2A).
Training induced a marked increase in muscle glycogen. This
effect was, however, only significant for Low. Muscle glyco-
gen was also measured in relation to two training sessions.
Muscle glycogen declined during the first bout of exercise.
Because the subjects were not allowed to eat in the recovery
period of the first bout of exercise, the second bout of exercise
undertaken by Low was initiated at a low muscle glycogen
level. Therefore, every second time the Low leg trained, it was
with a markedly low muscle glycogen level, whereas the High
leg initiated each training session with a high muscle glycogen
content (Fig. 2B).
Mitochondrial enzymes The activities of the mitochondrial
enzymes HAD and CS were measured in muscle biopsies
obtained at rest before and after 5 and 10 wk of training (Fig.
3). HAD activity increased with training, but only significantly
so in Low, whereas CS activity increased in both Low and
High. When the relative change from pretraining to after 10 wk
of training was estimated, there was a significantly more
pronounced increase in CS activity when Low was compared
with High.
Muscle fibers and capillaries. Percent number and percent
area of type IIX fibers decreased significantly in High, but
there was no difference between the two legs postexercise
(Table 3). There were no significant effects of training and no
difference between Low and High with regard to distribution of
fiber types or size or with regard to capillaries.
The main findings of the present study were that 1) that time
until exhaustion, 2) resting muscle glycogen concentration, and
3) CS activity were enhanced by training twice every second
day when compared with training once daily. The protocol
allowed us to compare the work performed in the prolonged
time trial with each leg at the same absolute as well as exercise
intensity. Using a study design where the two legs were trained
at different protocols further allowed us to distinguish possible
systemic and local effects. Thus systemic concentrations of, for
example, hormones and glucose were equal for the two legs,
and the study design therefore only measured possible local
differences as a consequence of different training schedules.
The present study was based on an overall hypothesis, which
can be expressed as follows: “Muscle glycogen: train low,
compete high.” This hypothesis refers to the fact that, whereas
numerous studies have demonstrated that low muscle glycogen
Table 1. Maximal power output and time until exhaustion at
90% of maximal power output before and after 10 wk of
training and total work before and after 10 wk of training
Pretraining Posttraining
Low High Low High
747776 1077* 1066
, min
5.00.7 5.61.2 19.72.4*
Total work, kJ 225257 11414*
Values are means SE. Low, leg trained with low muscle glycogen
protocol; High, leg trained with high muscle glycogen protocol; P
, maximal
power output; T
, time until exhaustion; total work, P
. *Significant
difference (P 0.05) from pretraining in Low. Significant difference (P
0.05) from pretraining, in High. Significant difference (P 0.05) between
Low and High..
Table 2. Hormone and lactate levels
Both Legs Low High
Pretraining Posttraining Pretraining Posttraining Pretraining Posttraining
Insulin 47.212.76 19.563.37* 28.298.60 17.867.81* 35.547.98 21.173.36*
Glucagon 90.115.61 137.4824.23* 74.338.37 129.5217.99* 75.688.69 109.5517.09*
Norepinephrine 2.10.25 5.790.82* 2.210.19 4.650.35* 2.110.26 3.370.36
Epinephrine 0.30.07 0.740.16* 0.280.05 0.890.18* 0.300.07 0.660.14
Cortisol 16.32.46 14.304.17 15.484.07 11.091.60 16.203.51 12.532.00
Lactate 1.10.37 3.280.66 1.130.13 3.110.70 1.380.11 2.080.34
Values are means SE. No significant differences were observed between pretraining values for Low and High legs. *Difference between pretraining and
posttraining; P 0.05.
Difference between Low and High legs posttraining value, P 0.05.
J Appl Physiol VOL 98 JANUARY 2005
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content is a limiting factor with regard to performance (6, 15,
28, 31, 36), this may not be valid when it comes to training
adaptation. In fact, data have accumulated showing that low
muscle glycogen content enhances the transcription and the
transcription rate of a number of genes involved in training
adaptation (22, 32, 42).
In the present study, the two legs were trained according to
different protocols: one leg performed one training session
Fig. 2. A: resting muscle glycogen concentration before (pre), halfway through
the training period (mid), and at the end (post) of the training period. Values are
geometric means SE. †Difference from pretraining level, P 0.05. B: muscle
glycogen content at rest, after one bout of two-legged training (Post 1st bout), and
after the subsequent bout of one-legged training (Post 2nd bout; with the Low leg).
The biopsies obtained after the 1st and 2nd bouts are from the Low leg only.
Values are geometric means SE. †Difference from pretraining level, P 0.05.
Fig. 3. A: resting muscle citrate synthase (CS) activity pretraining, midtrain-
ing, and posttraining. Values are means SE. †Difference from pretraining
level in Low, P 0.05. ‡Difference from pretraining level in High P 0.05.
B: resting muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity pretrain-
ing, midtraining, and posttraining. Values are means SE. †Difference from
pretraining level in Low, P 0.05. ‡Difference from pretraining level in High,
P 0.05. C: change in resting muscle CS and HAD activity from pretraining
to posttraining. Values are means SE. $Difference between Low and High,
P 0.05.
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daily, whereas the other leg performed two training sessions
separated by only 2 h every second day. The latter training
schedule resulted in a marked decrease in muscle glycogen
content after the first bout of exercise. Therefore, when the
second bout of exercise was performed within the same day, it
was undertaken with very low muscle glycogen content. Thus
we succeeded in developing a protocol that allowed us to
compare training at a low muscle glycogen with training at a
high muscle glycogen content. The finding that the catechol-
amine response to exercise performed at low muscle glycogen
was higher than at exercise performed at high muscle glycogen
concentration demonstrates that a higher stress response was
elicited when the muscle glycogen was low.
In the present study, resting muscle glycogen content in-
creased with training in accordance to numerous previous
studies (25). However, the increase in muscle glycogen was
only significant for Low. This indicates that training at the Low
protocol may be a more efficient training mode with respect to
enhancing muscle glycogen stores. It has long been known that
glycogen synthase (GS) activity is closely coupled to the
muscle glycogen content in both rodent (16, 17) and human
skeletal muscle (7), both in the resting state and after muscle
contraction (16, 38, 51). The rate-limiting conversion of UDP-
glucose to glycogen is catalyzed by GS, which in skeletal
muscle is known to be bound to glycogen particles (5, 35) and
myofibrils (33, 50). Rat studies have demonstrated that con-
traction-induced increase in GS activity is strongly dependent
on muscle glycogen (38). Exercise regulation of GS is charac-
terized by great complexity (39). GS is a substrate of kinases
and phosphatases acting on several phosphorylation sites of
GS, and exercise seems to activate both stimulatory and inhib-
itory regulators of GS, including activation of 5-AMP-acti-
vated protein kinase (11, 13, 40). The mechanisms responsible
for inhibition and especially activation are poorly understood.
It may be proposed that the GS activity during exercise may
depend on the relative strength of opposing signals. Glycogen
breakdown may be considered the major stimulatory signal.
The finding that training on the Low and High protocols
influence glycogen metabolism differently talks in favor of the
idea that glycogen breakdown and low muscle glycogen are
important stimulatory GS signals, which result in a total
increase in muscle glycogen concentration.
The effect on resting muscle glycogen does, however, not
explain the difference in “time until exhaustion” because this
test was carried out on a relatively high intensity, which did not
allow the volunteers to exercise for more than a maximum of
25 min. Therefore, muscle glycogen content was not a limiting
The study did not aim to measure peak muscle oxygen
consumption, and therefore we did not consider measuring the
rate-limiting enzyme -keto acid glutamate dehydrogenase (8).
Rather, we focused on citrate synthase as a more general
marker of the tricarboxylic acid cycle flux and HAD as the
most-used marker enzyme for the -oxidation.
The activity of the mitochondrial enzymes HAD and CS
increased with training in both Low and High. However,
regarding CS activity, this increase was more pronounced in
the leg that was trained in the low muscle glycogen protocol.
Transcriptional activities of HAD and CS are only markedly
influenced by acute muscle contractions (43).
However, the possibility exists that the mRNA for these
genes peak late in the recovery phase. The effect of low muscle
glycogen on HAD and CS gene activation has not been studied.
The finding that the low muscle glycogen protocol induced a
more pronounced enhancement of CS activity may represent
one mechanism explaining the enhanced endurance time in the
low muscle glycogen-trained leg.
We were unable to identify any major effects of training on
muscle fiber types, fiber size, or capillaries, although there was
the expected decrease in the amount of type 2X fibers in the
High leg, with a corresponding tendency in the Low leg.
Similarly, there was a clear, but not significant, tendency of an
increased capillary density in both legs. This lack of significant
adaptations in fiber types and capillary density is most likely
due to the major limitation of the present study: the training
protocol was demanding to such an extent that the cost limited
us to carry through only seven subjects. An n value of seven is
sufficient to study parameters with little variation, but can be a
major limitation in relation to adaptations in fiber types and
The present study should be viewed as one among hopefully
many studies to be conducted in the coming decade, investi-
gating the effect of muscle glycogen content on training adap-
tation using molecular biological methods as well as exercise
physiological parameters. Coaches and athletes should be care-
ful not to draw practical consequences of the present study with
regard to training regimens. In the real world, training on a
high muscle glycogen content may allow the athlete to train for
longer periods and thereby obtain better results. In addition,
training schedules that allow muscle glycogen to decrease to
low values may increase the risk for the so-called overtraining
syndrome (41).
In summary, in a human experimental laboratory setting,
training twice every second day was superior to training once
daily. The present study therefore suggests that perhaps some
adaptations to physical activity may require a cycling of
Table 3. Muscle fiber and capillary characteristics
pretraining and posttraining
Pretraining Posttraining
Low High Low High
Percent number
Type I 67.05.1 62.76.7 64.75.7 61.85.1
Type I/IIA 0.20.1 0.10.1 0.80.4 0.20.1
Type IIA 25.03.4 27.24.2 26.34.9 35.25.9
Type IIAX 5.02.1 6.52.0 7.53.8 2.31.0
Type IIX 2.41.3 3.21.1 0.40.3 0.30.3*
Percent area
Type I 65.25.0 58.86.3 63.76.3 61.86.6
Type I/IIA 0.20.1 0.00.0 0.80.4 0.20.1
Type IIA 28.84.3 31.84.9 27.95.3 35.27.2
Type IIAX 3.91.5 6.31.8 6.83.3 2.31.1
Type IIX 1.70.9 2.80.9 0.50.3 0.20.2*
Fiber size
Type I 4,963843 5,061893 5,216659 5,564781
Type II 5,394926 5,867676 5,319550 5,464558
Capillaries/type I
fiber 5.60.6 5.50.6 6.00.3 6.50.4
Capillaries/type II
fiber 5.30.7 5.70.5 5.70.4 6.10.5
55516 55237 59827 61033
Values are means SE. *Difference from pretraining, P 0.05.
J Appl Physiol VOL 98 JANUARY 2005
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muscle glycogen stores as recently suggested by Chakravarthy
and Booth (12).
We thank the subjects for participation. Ruth Rousing and Hanne Villumsen
are acknowledged for excellent technical assistance.
The study was also supported by grants from The Danish National Research
Foundation (no. 504-14), the Novo Nordisk Foundation, Lundbeckfonden,
Rigshospitalet, Member of H:S-Copenhagen Hospital. Civil Engineer Frode V.
Nyegaard og Hustrus Fond, Danfoss, Augustinus Fonden, Team Danmark, and
Kulturministeriets Udvalg for Idrætsforskning.
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... Hansen et al. (2005). The "Normal" leg completed daily exercise bouts with xiii high muscle glycogen, and the "low" leg completed 2 bouts of exercise every 2 days, with the first bout of exercise intended to deplete muscle glycogen before the following session. ...
... Principally, endurance exercise completed with reduced carbohydrate availability, or "train low", induces significant disturbance to the intra-muscular metabolic milieu, augmenting the molecular signalling responses associated with endurance training adaptation. Typically, the metabolic impact of commencing exercise with low carbohydrate availability is reduced muscle glycogenolytic rate (Arkinstall et al., 2004), increased circulating free fatty acid (FFA) availability and oxidation rates and elevated circulating catecholamines (Hansen et al., 2005). The resultant metabolic challenge invokes a compensatory adaptive response through the up-regulation of several molecular signalling processes responsible for orchestrating endurance training adaptation. ...
... The chronic application of "Train-low" provides positive performance outcomes and favourable alterations to body composition in trained individuals (Hansen et al., 2005;Yeo et al., 2008a;Marquet et al., 2016a;2016b), highlighting the practical benefit of reducing muscle glycogen concentration to promote exercise adaptation. ...
Endurance athletes have traditionally been advised to consume high carbohydrate intake before, during and after exercise to support high training loads and facilitate recovery. Accumulating evidence suggests periodically training with low carbohydrate availability, termed “train-low”, augments skeletal oxidative adaptations. Comparably, to account for increased carbohydrate utilisation during exercise in hot environmental conditions, nutritional guidelines advocate high carbohydrate intake. Recent evidence suggests heat stress induces oxidative adaptation in skeletal muscle, augmenting mitochondrial adaptation during endurance training. This thesis aimed to assess the efficacy of training with reduced carbohydrate and the impact of elevated ambient temperatures on performance and metabolism. Chapter 4 demonstrated 3 weeks of Sleep Low-Train Low (SL-TL) improves performance when prescribed and completed remotely. Chapter 5 implemented SL-TL in hot and temperate conditions, confirming SL-TL improves performance and substrate metabolism, whilst additional heat stress failed to enhance performance in hot and temperate conditions following the intervention. Chapters 6 and 7 optimised and implemented a novel in vitro skeletal muscle exercise model combining electrical pulse stimulation and heat stress. Metabolomics analysis revealed an ‘exercise-induced metabolic response, with no direct metabolomic impact of heat stress. Chapter 8 characterised the systemic metabolomic response to acute exercise in the heat following SL-TL and heat stress intervention revealing distinct metabolic signatures associated with exercise under heat stress. In summary, this thesis provides data supporting the application of the SL-TL strategy during endurance training to augment adaptation. Data also highlights the impact of exercise, environmental temperature and substrate availability on skeletal muscle metabolism and the systemic metabolome. Together, these data provide practical support for the efficacy of the SL-TL strategy to improve performance and adaptation whilst casting doubt on the utility of this approach in hot environments in endurance-trained athletes.
... Uygulanan bu yöntemi antrenörler gerçek hayata yansıtamamışlardır. Ek olarak bu çalışma antrenmansız kişilerde uygulanmıştır (Hansen et al., 2005). Daha sonra gerçek uygulanabilir bir yöntemle araştırma tekrar yapılmıştır. ...
... A decrease in skeletal muscle glycogen concentration triggers a cascade of intracellular signals that are associated with up-regulation of fatty acid metabolism and mitochondrial biogenesis in skeletal muscle, among other pathways (Hardie and Sakamoto, 2006;Philp et al., 2012;Pilegaard et al., 2002;Wojtaszewski et al., 2003). Training with low muscle glycogen has been attributed to improvements in skeletal muscle working of >2 fold capacity compared to training with normal muscle glycogen in humans after 10 weeks of training (Hansen et al., 2005). ...
Energy deficiency profoundly disrupts normal endocrinology, metabolism, and physiology, resulting in an orchestrated response for energy preservation. As such, despite energy deficit is typically thought as positive for weight-loss and treatment of cardiometabolic diseases during the current obesity pandemic, in the context of contemporary sports and exercise nutrition, chronic energy deficiency is associated to negative health and athletic performance consequences. However, the evidence of energy deficit negatively affecting physical capacity and sports performance is unclear. While severe energy deficiency can negatively affect physical capacity, humans can also improve aerobic fitness and strength while facing significant energy deficit. Many athletes, also, compete at an elite and world-class level despite showing clear signs of energy deficiency. Maintenance of high physical capacity despite the suppression of energetically demanding physiological traits seems paradoxical when an evolutionary viewpoint is not considered. Humans have evolved facing intermittent periods of food scarcity in their natural habitat and are able to thrive in it. In the current perspective it is argued that when facing limited energy availability, maintenance of locomotion and physical capacity are of high priority given that they are essential for food procurement for survival in the habitat where humans evolved. When energetic resources are limited, energy may be allocated to tasks essential for survival (e.g. locomotion) while minimising energy allocation to traits that are not (e.g. growth and reproduction). The current perspective provides a model of energy allocation during energy scarcity supported by observation of physiological and metabolic responses that are congruent with this paradigm.
... It is well known that increasing muscle glycogen level with carbohydrate loading improves endurance performance. Moreover, a new glycogen strategy known as "train low" has been developed in recent years [4,5]. The typical procedure of train low involves the depletion of 40-50% of glycogen levels by prior training, and subsequent training is conducted at a low glycogen state [6]. ...
Full-text available
Muscle glycogen is a crucial energy source for exercise, and assessment of muscle glycogen storage contributes to the adequate manipulation of muscle glycogen levels in athletes before and after training and competition. Muscle biopsy is the traditional and gold standard method for measuring muscle glycogen; alternatively, 13C magnetic resonance spectroscopy (MRS) has been developed as a reliable and non-invasive method. Furthermore, outcomes of ultrasound and bioimpedance methods have been reported to change in association with muscle glycogen conditions. The physiological mechanisms underlying this activity are assumed to involve a change in water content bound to glycogen; however, the relationship between body water and stored muscle glycogen is inconclusive. In this review, we discuss currently available muscle glycogen assessment methods, focusing on 13C MRS. In addition, we consider the involvement of muscle glycogen in changes in body water content and discuss the feasibility of ultrasound and bioimpedance outcomes as indicators of muscle glycogen levels. In relation to changes in body water content associated with muscle glycogen, this review broadens the discussion on changes in body weight and body components other than body water, including fat, during carbohydrate loading. From these discussions, we highlight practical issues regarding muscle glycogen assessment and manipulation in the sports field.
... Exercise is widely known to increase muscle glycogen storage and glycogen metabolism (Hargreaves 1997;Hansen et al. 2005). This generally holds true in fish (Jobling et al. 1993) and was generally reaffirmed in the current study. ...
Full-text available
Exercise has been shown to increase growth of many salmonid species. However, limited research has evaluated exercise on warmwater species. The present study was conducted to evaluate with tilapia, red drum (RD), and hybrid striped bass (HSB), the effects of swimming (exercising) in a constant slow current of approximately one body length/s (1bl/s) compared to not being forced to swim in a static culture system. Concurrent trials were conducted with 22 advanced juvenile male Nile tilapia (Wt0 97.9 ± 2.4 g), 38 juvenile red drum (Wt0 74.9 ± 4.4 g), and 20 juvenile HSB (Wt0 78.0 ± 3.2 g). Equal numbers of fish of each species were pit tagged and randomly assigned to two tanks, one operated static (control) and the other with current (exercised), which were all part of the same recirculating aquaculture system. Fish were fed to satiation twice daily a commercial diet and individually weighed every 2 weeks through 7 weeks. Significant (P ≤ 0.05) enhancements of weight gain were observed for exercised tilapia and RD vs static (control) treatments. Reduced growth was observed in exercised HSB, possibly due to consistently skittish feeding behavior. Hepatosomatic index was lower in all exercised fish, though not significantly so for RD and tilapia. Significant reductions also were detected in liver glycogen of exercised tilapia and RD. Results from this study indicate that continuous exercise beneficially affected aspects of tilapia and red drum growth and altered their body composition.
... As total energy requirements and, consequently, carbohydrate demands are high in endurance-based sports, it is fair to assume that optimization of carbohydrate intake in these sport disciplines plays an important role. Early sports nutrition guidelines [131] advised athletes to both train and compete with high carbohydrate availability, and this approach dominated until 2005, when Hansen and colleagues observed that a reduction in carbohydrate availability before certain training sessions in untrained individuals could potentially enhance training adaptations [132]. In this study, leg kicking exercise training was performed in a 10-week-long training study. ...
Full-text available
The importance of carbohydrate as a fuel source for exercise and athletic performance is well established. Equally well developed are dietary carbohydrate intake guidelines for endurance athletes seeking to optimize their performance. This narrative review provides a contemporary perspective on research into the role of, and application of, carbohydrate in the diet of endurance athletes. The review discusses how recommendations could become increasingly refined and what future research would further our understanding of how to optimize dietary carbohydrate intake to positively impact endurance performance. High carbohydrate availability for prolonged intense exercise and competition performance remains a priority. Recent advances have been made on the recommended type and quantity of carbohydrates to be ingested before, during and after intense exercise bouts. Whilst reducing carbohydrate availability around selected exercise bouts to augment metabolic adaptations to training is now widely recommended, a contemporary view of the so-called train-low approach based on the totality of the current evidence suggests limited utility for enhancing performance benefits from training. Nonetheless, such studies have focused importance on periodizing carbohydrate intake based on, among other factors, the goal and demand of training or competition. This calls for a much more personalized approach to carbohydrate recommendations that could be further supported through future research and technological innovation (e.g., continuous glucose monitoring). Despite more than a century of investigations into carbohydrate nutrition, exercise metabolism and endurance performance, there are numerous new important discoveries, both from an applied and mechanistic perspective, on the horizon.
Endurance exercise performance is known to be closely associated with the three physiological pillars of maximal O2 uptake ( V ̇ O 2 max $\dot{V}_{{\rm O}_{2}{\rm max}}$ ), economy or efficiency during submaximal exercise, and the fractional utilisation of V ̇ O 2 max $\dot{V}_{{\rm O}_{2}{\rm max}}$ (linked to metabolic/lactate threshold phenomena). However, while 'start line' values of these variables are collectively useful in predicting performance in endurance events such as the marathon, it is not widely appreciated that these variables are not static but are prone to significant deterioration as fatiguing endurance exercise proceeds. For example, the 'critical power' (CP), which is a composite of the highest achievable steady-state oxidative metabolic rate and efficiency (O2 cost per watt), may fall by an average of 10% following 2 h of heavy intensity cycle exercise. Even more striking is that the extent of this deterioration displays appreciable inter-individual variability, with changes in CP ranging from <1% to ∼32%. The mechanistic basis for such differences in fatigue resistance or 'physiological resilience' are not resolved. However, resilience may be important in explaining superlative endurance performance and it has implications for the physiological evaluation of athletes and the design of interventions to enhance performance. This article presents new information concerning the dynamic plasticity of the three 'traditional' physiological variables and argues that physiological resilience should be considered as an additional component, or fourth dimension, in models of endurance exercise performance.
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Human skeletal muscle demonstrates remarkable plasticity, adapting to numerous external stimuli including the habitual level of contractile loading. Accordingly, muscle function and exercise capacity encompass a broad spectrum, from inactive individuals with low levels of endurance and strength, to elite athletes who produce prodigious performances underpinned by pleiotropic training-induced muscular adaptations. Our current understanding of the signal integration, interpretation and output coordination of the cellular and molecular mechanisms that govern muscle plasticity across this continuum is incomplete. As such, training methods and their application to elite athletes largely rely on a "trial and error" approach with the experience and practices of successful coaches and athletes often providing the bases for "post hoc" scientific enquiry and research. This review provides a synopsis of the morphological and functional changes along with the molecular mechanisms underlying exercise adaptation to endurance- and resistance-based training. These traits are placed in the context of innate genetic and inter-individual differences in exercise capacity and performance, with special considerations given to the ageing athletes. Collectively, we provide a comprehensive overview of skeletal muscle plasticity in response to different modes of exercise, and how such adaptations translate from "molecules to medals".
Acute exercise increases liver gluconeogenesis to supply glucose to working muscle. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of gluconeogenesis. This functional coupling between liver gluconeogenesis and lipid oxidation has been proposed to underlie the ability of regular exercise to enhance liver mitochondrial oxidative metabolism and decrease liver steatosis in individuals with non-alcoholic fatty liver disease. Herein we tested whether repeated bouts of increased hepatic gluconeogenesis are necessary for exercise training to lower liver lipids. Experiments used diet-induced obese mice lacking hepatic phosphoenolpyruvate carboxykinase 1 (KO) to inhibit gluconeogenesis and wild type (WT) littermates. ² H/ ¹³ C metabolic flux analysis quantified glucose and mitochondrial oxidative fluxes in untrained mice at rest and during acute exercise. Circulating and tissue metabolite levels were determined during sedentary conditions, acute exercise, and refeeding post-exercise. Mice also underwent six weeks of treadmill running protocols to define hepatic and extrahepatic adaptations to exercise training. Untrained KO mice were unable to maintain euglycemia during acute exercise resulting from an inability to increase gluconeogenesis. Liver triacylglycerides were elevated following acute exercise and circulating β-hydroxybutyrate was higher during post-exercise refeeding in untrained KO mice. In contrast, exercise training prevented liver triacylglyceride accumulation in KO mice. This was accompanied by pronounced increases in indices of skeletal muscle mitochondrial oxidative metabolism in KO mice. Together, these results show that hepatic gluconeogenesis is dispensable for exercise training to reduce liver lipids. This may be due to responses in ketone body metabolism and/or metabolic adaptations in skeletal muscle to exercise.
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A fraction containing a protein-glycogen complex was obtained from rabbit muscle by different procedures involving acid precipitation followed by differential centrifugation, direct differential centrifugation, and acetone fractionation. All three preparations were essentially identical in terms of their physical, chemical, and enzymatic characteristics strongly suggesting that the protein-glycogen complex represents a structural and functional unit of the cell rather than artifacts resulting from a given isolation procedure. Electron micrographs of the isolated fractions showed the presence of glycogen granules together with vesicles arising from fragments of the sarcoplasmic reticulum. Sedimentation velocity patterns obtained in the analytical ultracentrifuge, sucrose gradient centrifugation, and Sepharose 2B gel filtration indicated that the material can be separated into a light (approximately 120 S) and a heavy (approximately 600 S) fraction. The light fraction consists mainly of glycogen particles to which are associated phosphorylase, phosphorylase kinase, and phosphatase among other enzymes. The heavy fraction contains mainly the elements of the sarcoplasmic reticulum characterized by a strong ATPase activity and some lysosomes as indicated by the presence of traces of acid phosphatase and amylase activity. Contamination of the glycogen particles by this latter enzyme resulted in their slow degradation that prevented further purification.
Protein dephosphorylation by phosphatase PP1 plays a central role in mediating the effects of insulin on glucose and lipid metabolism. A PP1C-targeting protein expressed in 3T3-L1 adipocytes (called PTG, for protein targeting to glycogen) was cloned and characterized. PTG was expressed predominantly in insulin-sensitive tissues. In addition to binding and localizing PP1C to glycogen, PTG formed complexes with phosphorylase kinase, phosphorylase a, and glycogen synthase, the primary enzymes involved in the hormonal regulation of glycogen metabolism. Overexpression of PTG markedly increased basal and insulin-stimulated glycogen synthesis in Chinese hamster ovary cells overexpressing the insulin receptor, which do not express endogenous PTG. These results suggest that PTG is critical for glycogen metabolism, possibly functioning as a molecular scaffold.
• The influence of muscle glycogen content on glycogen synthase (GS) localization and GS activity was investigated in skeletal muscle from male Wistar rats. • Two groups of rats were obtained, preconditioned with a combination of exercise and diet to obtain either high (HG) or low (LG) muscle glycogen content. The cellular distribution of GS was studied using subcellular fractionation and confocal microscopy of immunostained single muscle fibres. Stimulation of GS activity in HG and LG muscle was obtained with insulin or contractions in the perfused rat hindlimb model. • We demonstrate that GS translocates from a glycogen-enriched membrane fraction to a cytoskeleton fraction when glycogen levels are decreased. Confocal microscopy supports the biochemical observations that the subcellular localization of GS is influenced by muscle glycogen content. GS was not found in the nucleus. • Investigation of the effect of glycogen content on GS activity in basal and insulin- and contraction-stimulated muscle shows that glycogen has a strong inhibitory effect on GS activity. Our data demonstrate that glycogen is a more potent regulator of glycogen synthase activity than insulin. Furthermore we show that the contraction-induced increase in GS activity is merely a result of a decrease in muscle glycogen content. • In conclusion, the present study shows that GS localization is influenced by muscle glycogen content and that not only basal but also insulin- and contraction-stimulated GS activity is strongly regulated by glycogen content in skeletal muscle.
The muscle glycogen content of the quadriceps femoris muscle was determined in 9 healthy subjects with the aid of the needle biopsy technique. The glycogen content could be varied in the individual subjects by instituting different diets after exhaustion of the glycogen store by hard exercise. Thus, the glycogen content after a fat ± protein (P) and a carbohydrate-rich (C) diet varied maximally from 0.6 g/100g muscle to 4.7 g. In all subjects, the glycogen content after the C diet was higher than the normal range for muscle glycogen, determined after the mixed (M) diet. After each diet period, the subjects worked on a bicycle ergometer at a work load corresponding to 75 per cent of their maximal O2 uptake, to complete exhaustion. The average work time was 59, 126 and 189 min after diets P, M and C, and a good correlation was noted between work time and the initial muscle glycogen content. The total carbohydrate utilization during the work periods (54–798 g) was well correlated to the decrease in glycogen content. It is therefore concluded that the glycogen content of the working muscle is a determinant for the capacity to perform long-term heavy exercise. Moreover, it has been shown that the glycogen content and, consequently, the long-term work capacity can be appreciably varied by instituting different diets after glycogen depletion.
The results indicate a gradual conversion of a part of the type IIB fiber population into type IIA fibers in response to an endurance training program. The pretraining percentage distribution of type IIA and IIB fibers was in the same range as that reported by Hedberg and Jansson (1976) for 68 sixteen year old boys (32% IIA and 13% IIB fibers). It is worth emphasizing that the classification of the type II fibers into the subgroups IIA and IIB in this study is based on differences in the pH sensitivity of the myosin ATPase. According to Brooke and Kaiser (1970) this difference is related to the reactivity of sulfhydryl groups of the myosin molecule. Thus it is conceivable that the observed changes indeed indicate a change in the structure of the myosin molecule.
• Ten subjects performed incremental exercise up to their maximum work rate with the knee extensors of one leg. Measurements of leg blood flow and femoral arteriovenous differences of oxygen were made in order to be able to calculate oxygen uptake of the leg. • The volume of the quadriceps muscle was determined from twenty-one to twenty-five computer tomography section images taken from the patella to the anterior inferior iliac spine of each subject. • The maximal activities of three enzymes in the Krebs cycle, citrate synthase, oxoglutarate dehydrogenase and succinate dehydrogenase, were measured in biopsy samples taken from the vastus lateralis muscle. • The average rate of oxygen uptake over the quadriceps muscle at maximal work, 353 ml min−1kg−1, corresponded to a Krebs cycle rate of 4.6 mol min−1 g−1. This was similar to the maximal activity of oxoglutarate dehydrogenase (5.1 mol min−1 g−1), whereas the activities of succinate dehydrogenase and citrate synthase averaged 7.2 and 48.0 mol min−1 g−1, respectively. • It is suggested that of these enzymes, only the maximum activity of oxoglutarate dehydrogenase can provide a quantitative measure of the capacity of oxidative metabolism, and it appears that the enzyme is fully activated during one-legged knee extension exercise at the maximal work rate.
This study compared the effects of supplementing the normal diets of six trained cyclists [maximal oxygen uptake O2max) 4.5 (0.36)l · min−1; values are mean (SD)] with additional carbohydrate (CHO) on muscle glycogen utilisation during a 1-h cycle time-trial (TT). Using a randomised crossover design, subjects consumed either their normal diet (NORM) for 3 days, which consisted of 426 (137) g · day−1 CHO [5.9 (1.4) g · kg−1 body mass (BM)], or additional CHO (SUPP) to increase their intake to 661 (76) g · day−1 [9.3 (0.7) g · kg−1 BM]. The SUPP diet elevated muscle glycogen content from 459 (83) to 565 (62) mmol · kg−1 dry weight (d.w.) (P < 0.05). However, despite the increased pre-exercise muscle glycogen stores, there was no difference in the distance cycled during the TT [40.41 (1.44) vs 40.18 (1.76) km for NORM and SUPP, respectively]. With NORM, muscle glycogen declined from 459 (83) to 175 (64) mmol · kg−1 d.w., whereas with SUPP the corresponding values were 565 (62) and 292 (113) mmol · kg−1 d.w. Accordingly, both muscle glycogen utilisation [277 (64) vs 273 (114) mmol · kg−1 d.w.] and total CHO oxidation [169 (20) vs 165 (30) g · h−1 for NORM and SUPP, respectively] were similar. Neither were there any differences in plasma glucose or lactate concentrations during the two experimental trials. Plasma glucose concentration averaged 5.5 (0.5) and 5.6 (0.6) mmol · l−1, while plasma lactate concentration averaged 4.4 (1.9) and 4.4 (2.3) mmol · l−1 for NORM and SUPP, respectively. The results of this study show that when well-trained subjects increase the CHO content of their diet for 3 days from 6 to 9 g · kg−1 BM there is only a modest increase in muscle glycogen content. Since supplementary CHO did not improve TT performance, we conclude that additional CHO provides no benefit to performance for athletes who compete in intense, continuous events lasting 1 h. Furthermore, the substantial muscle CHO reserves observed at the termination of exercise indicate that whole-muscle glycogen depletion does not determine fatigue at this exercise intensity and duration.