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Effects of Dehydration during Cycling on
Skeletal Muscle Metabolism in Females
HEATHER M. LOGAN-SPRENGER
1
, GEORGE J. F. HEIGENHAUSER
2
, KIERAN J. KILLIAN
2
, and
LAWRENCE L. SPRIET
1
1
Department of Human Health and Nutritional Sciences, University of Guelph, Ontario, CANADA;
and
2
Department of Medicine, McMaster University, Hamilton, Ontario, CANADA
ABSTRACT
LOGAN-SPRENGER, H. M., G. J. F. HEIGENHAUSER, K. J. KILLIAN, and L. L. SPRIET. Effects of Dehydration during Cycling on
Skeletal Muscle Metabolism in Females. Med. Sci. Sports Exerc., Vol. 44, No. 10, pp. 1949–1957, 2012. Introduction: This study
investigated the effects of progressive dehydration on the time course of changes to whole body substrate oxidation and skeletal muscle
metabolism during 120 min of cycling in hydrated females. Methods: Subjects (n= 9) cycled for 120 min at approximately 65% V
˙O
2peak
on two occasions: with no fluid (DEH) and with fluid (HYD) replacement to match sweat losses. Venous blood samples were taken at
rest and every 20 min and muscle biopsies taken at 0, 60, and 120 min of exercise. Results: DEH subjects lost 0.9% body mass from 0 to
60 min and 1.1% from 60 to 120 min (2.0% total). HR and core temperature (Tc) were significantly greater from 30 to 120 min, plasma
volume (Pvol) loss from 40 to 120 min, and RPE from 60 to 120 min in the DEH trial. There were no differences in V
˙O
2
or sweat loss
between trials. RER (HYD, 0.85 T0.01, vs. DEH, 0.87 T0.01) and total CHO oxidation (175 T17 vs. 191 T17 g) were higher in the DEH
trial. Blood (La) was significantly higher in the DEH trial, with no change in plasma free fatty acid and epinephrine concentrations.
Muscle glycogenolysis was 31% greater in the DEH trial (252 T49 vs. 330 T33 mmolIkg
j1
dry muscle), and muscle (La) was also higher
at 60 min. Conclusion: Progressive dehydration significantly increased HR, Tc, RPE, Pvol loss, whole body CHO oxidation, and muscle
glycogenolysis, and these changes were already apparent in the first hour of exercise when body mass losses were e1%. The increased
muscle glycogenolysis with DEH appeared to be due to increased core and muscle temperature, secondary to less efficient movement
of heat from the core to the periphery. Key Words: HYDRATION, EXERCISE, FLUID INTAKE, BODY MASS LOSS, SWEAT
RATE, SUBSTRATE OXIDATION
Mild dehydration during exercise can be a major
concern for athletes because it may lead to pre-
mature fatigue. The cardiovascular and thermo-
regulatory consequences of exercising dehydrated are well
documented with the magnitude of these effects directly
proportional to the degree of dehydration (2,14,21,23,25,27,
28,30,34,36,38,39). As little as a 2% loss in body mass (BM)
due to dehydration has been consistently reported to result
in elevated HR, core temperature (Tc), rate of perceived ex-
ertion, and plasma osmolality. However, little research has
investigated the effects of exercise-induced dehydration on
whole body substrate use and skeletal muscle metabolism.
Hargreaves et al. (17) investigated the effects of exercise-
induced dehydration on muscle metabolism in males in a
temperate environment (20-C–22-C) and reported that a 3%
BM loss resulted in a significantly higher rectal and muscle
temperature (Tm) at 120 min of exercise, with no difference
in rectal temperature between trials at any other time point
during exercise. The respiratory exchange ratio was also sig-
nificantly higher in the fluid-restricted trial after 60 and
120 min of exercise, with the difference between trials being
greater in the second hour of cycling. The study reported a
16% greater glycogen use during the 120 min of exercise
(17). Other studies reported similar findings but have been
performed in the heat. For example, Gonzalez-Alonso et al.
(15) had seven males cycle until volitional exhaustion (135 T
4 min) in the heat (35-C) while developing progressive de-
hydration to approximately 3.9% BM loss. They reported
increased carbohydrate oxidation, muscle glycogen use (45%
greater), muscle lactate accumulation, and net lactate release
across the contracting leg compared with being euhydrated.
More recently, Febbraio (9) reviewed the relevant literature
examining exercise in the heat and concluded that when Tm
is higher than control during exercise, there is augmented
muscle glycogenolysis. However, some studies did not con-
trol for hydration status and were all conducted in males.
Presently, there are no studies investigating the time course
of progressive exercise-induced dehydration on whole body
substrate oxidation and skeletal muscle metabolismin females.
Address for correspondence: Heather M. Logan-Sprenger, Ph.D., Depart-
ment of Human Health and Nutritional Sciences, University of Guelph,
Ontario N1G 2W1, Canada; E-mail: hlogan@uoguelph.ca.
Submitted for publication January 2012.
Accepted for publication April 2012.
0195-9131/12/4410-1949/0
MEDICINE & SCIENCE IN SPORTS & EXERCISE
Ò
Copyright Ó2012 by the American College of Sports Medicine
DOI: 10.1249/MSS.0b013e31825abc7c
1949
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Copyright © 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
It has been suggested that women thermoregulate less
effectively because of higher Tc during the same load ex-
ercise compared with males (26,40). Work by Gagnon et al.
(13) also suggests that females experience a quicker rise in
Tc during exercise, which may accelerate muscle glycogen
use. The effect of the heightened Tc during prolonged ex-
ercise in females coupled with the thermal stress associated
with progressive dehydration on substrate oxidation and mus-
cle metabolism has yet to be elucidated.
In light of the small amount of information examining the
effects of mild dehydration (1%–2%) on muscle metabolism
in general, we investigated the effects of progressive exercise-
induced dehydration in hydrated females to determine the
time course of changes to physiological responses and skel-
etal muscle metabolism. We hypothesized that because fe-
male subjects progressively dehydrated during exercise and
Tc increased above the increase in the hydrated state, there
would be a greater reliance on whole body carbohydrate
oxidation and muscle glycogenolysis during prolonged ex-
ercise. As well, we expected that these differences would
be augmented in the second hour of exercise in the dehy-
drated trial.
METHODS
Subjects. Nine recreationally active females (mean age,
21.7 T0.6 yr; height, 155.7 T2.8 cm; weight, 58.8 T2.8 kg;
and V
˙O
2peak
, 2.9 T0.2 LImin
j1
, participated in the study. All
subjects engaged in recreational physical activity 2–3 dIwk
j1
and were taking oral contraceptives. Testing occurred at a
time during their menstrual cycle other than ovulation. Sub-
jects were informed both verbally and in writing of the ex-
perimental protocol and potential risks before giving their
written consent to participate. The Research Ethics Boards
of the University of Guelph and McMaster University ap-
proved the study.
STUDY DESIGN
Preexperimental protocol. In preparation for the ex-
periment, subjects visited the laboratory on three separate
occasions. On the first visit, subjects performed an incre-
mental cycling test to exhaustion on an electronically braked
cycle ergometer (LODE Excalibur; Quinton Instrument,
Groningen, The Netherlands) for the determination of V
˙O
2peak
.
Respiratory gases were collected and analyzed using a met-
abolic cart (MOXUS metabolic system; AEI Technologies,
Pittsburgh, PA). After a 30-min break, subjects cycled for
approximately 20 min at approximately 65% V
˙O
2peak
to es-
tablish the power output for the subsequent 120-min trials.
On two subsequent occasions, subjects reported to the lab-
oratory for practice trials and cycled at approximately 65%
V
˙O
2peak
for 120 min without fluid (DEH) or with fluid (HYD)
to replace sweat losses. DEH trials occurred first to ascer-
tain sweat losses over the 120-min trial and determine how
much fluid subjects needed to drink throughout the HYD
trial to maintain fluid balance. All subjects abstained from
strenuous exercise and caffeine and recorded their diet in
the 24 h before the trials. Two hours before the practice
rides, subjects ingested a meal provided for them (790 kcal;
144 g of carbohydrate, 35 g of fat, and 19 g of protein) and
250 mL of fluid. Subjects also drank 300 mL of water 90
and 45 min before each trial to ensure they were well hy-
drated before cycling. Upon arrival to the laboratory, sub-
jects voided their bladder and provided a small midstream
urine sample to determine urine specific gravity (USG) and
completely voided their bladder. A pretrial BM measurement
was made wearing only dry shorts and a sports bra. After 60
and 120 min of exercise, subjects stopped cycling and dis-
mounted the cycle ergometer, removed their shoes and shirt,
toweled dry, and were weighed wearing only shorts and a
sports bra for the determination of sweat loss during the
previous hour of exercise. At 60 min, subjects put on a dry
T-shirt and recommenced cycling. Any urine produced was
collected during each trial to account for total sweat loss
using the following equation:
sweat loss = (pre-BM jpost-BM (kg))
+ fluid intake (mL)
jurine loss (mL)
Three-minute respiratory gas measurements were col-
lected every 20 min during exercise to determine the volume
of oxygen consumed (V
˙O
2
), to determine the volume of
carbon dioxide produced (V
˙CO
2
), and to calculate the res-
piratory exchange ratio (29) and whole body CHO and fat
oxidation with the use of the nonprotein RER table and the
following equations:
CHO oxidation (g) = 4.585 (V
˙CO
2
)j3.226 (V
˙O
2
)
and
fat oxidation (g) = 1.695 (V
˙O
2
)j1.701 (V
˙CO
2
) (12,29).
Practice trials were separated by 5–7 d.
Experimental protocol. Subjects arrived to the labo-
ratory on two occasions for the actual experiment. During
the experimental trials, subjects cycled at approximately 65%
V
˙O
2peak
for 120 min with fluid to match sweat losses (HYD)
or without fluid (DEH). Subjects replicated the same pro-
cedure as described above for the practice trials. In addi-
tion, HR was collected using a Polar RS400 downloadable
HR monitor (Polar Electro, Lachine, QC), and Tc was de-
termined using an individually calibrated ingestible therm-
istor (HQ Inc., Palmetto, FL) that was ingested 3–5 h before
each trial. Before exercise, a Teflon catheter was inserted
into an antecubital vein for blood sampling and was flushed
with 0.9% saline to maintain patency. One leg was also pre-
pared for percutaneous needle biopsy sampling of the vastus
lateralis muscle by the Bergstro¨m technique (4). Three in-
cisions were made in the skin and deep fascia under local
anesthesia (2% xylocaine without epinephrine (EPI)) for
three separatebiopsies. Immediately before exercise, a venous
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blood (approximately 5 mL) sample and a muscle biopsy
were obtained while the subject rested on a bed. All muscle
samples were immediately frozen in the needle in liquid ni-
trogen and stored in liquid nitrogen for subsequent analyses.
Subjects then cycled for 120 min at approximately 65%
V
˙O
2peak
at a constant cadence (80–95 rpm). Venous blood
samples were obtained at 20, 40, 60, 80, 100, and 120 min
of exercise. HR, Tc, and RPE were recorded every 15 min
during exercise. RPE was determined using the Borg scale
(rating, 6–20) (5). During the HYD trial, subjects were given
fluid every 15 min to match sweat loss and drank the fluid
after HR, Tc, and RPE measurements were recorded. At 60
and 120 min, the subject stopped cycling and a muscle biopsy
was taken with the subject sitting on the cycle ergometer.
After the muscle biopsy was taken, subjects removed their
shoes and shirt, toweled dry, and were weighed for the de-
termination of BM loss over the previous 60 min of exercise.
The same procedure was replicated for the second trial, with
muscle biopsies taken from the opposite leg, and the trials
were randomized and separated by 7 d.
ANALYSES
Trial conditions. Laboratory temperature (-C) and rel-
ative humidity (%) were measured using a digital thermom-
eter (Fisher Scientific, Ottawa, ON). USG was measured via
a hand-held pocket refractometer (Model PAL-10S; ATAGO
USA Inc., Bellevue, WA) to assess hydration status from the
preexercise urine sample. The refractometer was calibrated
with distilled water before each measurement. Stover et al.
(35) reported that USG measured with refractometry strongly
correlated with urine osmolality (r= 0.995), and a USG
of 1.020 correlated with urine osmolality of approximately
800 mOsmIkg
j1
. In light of this and the published position
stand from the American College of Sports Medicine, a USG
below 1.020 was considered to indicate a hydrated state (30).
Blood measurements. Venous blood was collected in
sodium heparin tubes. A portion of whole blood (200 KL)
was added to 1 mL of 0.6 M perchloric acid and centrifuged.
The supernatant was stored at j20-C and later analyzed for
blood glucose and lactate with fluorometric techniques (3).
A second portion (1.5 mL) was centrifuged, and the super-
natant was analyzed for plasma free fatty acids (FFAs) with
an enzymatic colorimetric technique (NEFA C test kit; Wako
Chemicals, Richmond, VA). A third portion (1.5 mL) was
added to 30 mL of ethylene glycol tetraacetic acid and re-
duced glutathione and centrifuged (10,000g) for 3 min, and
the supernatant was analyzed for EPI with an enzymatic
immunoassay kit (EPI RIA kit; Rocky Mountain Diag-
nostics Inc., Colorado Springs, CO). The remaining venous
blood was used for the determination of whole blood he-
moglobin (Hb) and hematocrit (Hct). Hb was measured in
duplicate using an automated blood analysis machine
(OSM3 Hemoximeter; Radiometer, Copenhagen, Denmark).
Hct was measured in triplicate using capillary tubes and a
micro-Hct centrifuge and reader (microcapillary reader;
Damon/IEC Division, Needham Heights, MA). The per-
centage plasma volume change (%Pvol) was calculated using
whole blood Hb and Hct measurements (7).
Muscle metabolites. Each muscle biopsy was freeze
dried, powdered, and dissected free of visible connective tissue,
fat, and blood. One aliquot of freeze-dried powdered muscle
(approximately 10 mg) was extracted in 0.5 M HClO
4
–1 mM
EDTA and neutralized with 2.2 M KHCO
3
. The supernatant
was used to measure phosphocreatine (PCr), creatine (Cr),
adenosine triphosphate (ATP), and lactate. Muscle metabo-
lites were normalized to the highest total Cr content mea-
sured from all biopsies from each subject. Muscle glycogen
content was determined in duplicate using two additional
aliquots of freeze-dried muscle (2–4 mg). Glycogen was
extracted in 0.1 M NaOH and neutralized with 0.1 M HCl–
0.2 M citric acid–0.2 M Na
2
PO
4
, and amyloglucosidase was
added to degrade glycogen to glucose, which was measured
spectrophotometrically and normalized for total Cr (3).
Muscle calculations. Free adenosine diphosphate (ADPf )
and free AMP (AMPf ) contents were calculated by assum-
ing equilibrium of the Cr kinase and adenylate kinase reac-
tions (8). Specifically, ADPf was calculated using the measured
ATP, Cr, and PCr values, an estimated H
+
concentration,
and the Cr kinase constant of 1.66 109 (26). AMPf was
calculated from the estimated ADPf and measured ATP con-
tent using the adenylate kinase equilibrium constant of 1.05.
STATISTICAL ANALYSIS
All data were tested for normality of distribution and pre-
sented as the mean TSE. Time versus trial data were as-
sessed using a two-way ANOVA, and specific differences
were located using the Student–Newman–Keuls post hoc
test. A paired t-test was used to compare single parameter
data between trials. Statistical significance was accepted as
PG0.05.
RESULTS
Trial conditions. No significant pretrial differences
existed between the HYD and DEH trials for laboratory
TABLE 1. Differences in sweat loss, BM, and percentage BM loss between the dehydrated (DEH) and hydrated (HYD) trial at 0, 60, and 120 min of cycling at approximately 65% V
˙O
2peak
.
HYD DEH
Time (min) 0 60 120 0 60 120
Sweat loss (L) 0.8 T0.1 0.5 T0.1 0.7 T0.1 0.6 T0.1
BM (kg) 58.9 T2.9 59.0 T2.9 58.8 T2.9 58.8 T2.8 58.3 T2.8* 57.6 T2.7*
Percentage BM loss —– —– 0.9% T0.1% 2.0% T0.2%
Values are means TSE (n= 9).
* Significantly lower than HYD and 0 min in DEH (PG0.05).
EFFECTS OF DEHYDRATION ON MUSCLE METABOLISM Medicine & Science in Sports & Exercise
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temperature (HYD, 22.7-CT0.1-C, vs. DEH, 22.5-CT
0.1-C), relative humidity (34% T2.9% vs. 31% T3.4%),
pretrial BM (58.9 T2.9 vs. 58.8 T2.8 kg), or hydration state
(USG, 1.007 T0.002 vs. 1.010 T0.003).
BM loss, sweat loss, and fluid intake. BM was
maintained in the HYD trial by drinking a mean of 1.2 T
0.9 L of fluid over 120 min of cycling (Table 1). In the
DEH trial, BM was significantly lower at 60 and 120 min of
cycling, resulting in BM losses of 0.9% and 2.0% (Table 1).
There were no significant differences in sweat loss between
the HYD and DEH trials (Table 1). Only two of nine sub-
jects produced urine after the HYD trial (590 and 250 mL)
and only one subject after the DEH trial (160 mL).
Oxygen uptake and whole body substrate use. Mean
V
˙O
2
increased in both trials with exercise time and was signif-
icantly greater than 20 min at 40, 60, 80, 100, and 120 min in
both trials. There was no difference in V
˙O
2
between tri-
als (Fig. 1A). The RER progressively decreased in both
trials over time and was significantly lower than 20 min at
all time points in each trial (Fig. 1B). As well, the RER was
significantly higher in the DEH versus HYD trial from 40 to
120 min. CHO oxidation from 0 to 60 and 60 to 120 min
was significantly greater in the DEH (0–60 min, 111 T7g;
60–120 min, 82 T7 g) vs. HYD trial (102 T7 and 73 T5 g),
and fat oxidation was significantly lower from 0 to 60 and
60 to 120 min in the DEH (0–60 min, 17 T4 g; 60–120 min,
30 T3 g) versus HYD trial (21 T4 and 35 T3 g). Total CHO
oxidation was significantly greater in the DEH (193 T17 g)
versus HYD (175 T17 g) trials, and total fat oxidation was
FIGURE 1—The effect of fluid intake on V
˙O
2
(A) and respiratory ex-
change ratio (B) during 120 min of cycling at approximately 65% V
˙O
2peak
in the hydrated (HYD) and dehydrated (DEH) trials. Data are means T
SE (n=9).V
˙O
2
and RER were significantly greater than 20 min at all
time points in both trials (PG0.05). *Significantly greater than HYD trial
(PG0.05). Arrows (,) indicate approximately 1% and 2% BM loss.
FIGURE 2—The effect of fluid intake on HR (A), Tc (B), and RPE (C)
during 120 min of cycling at approximately 65% V
˙O
2peak
in the hy-
drated (HYD) and dehydrated (DEH) trials. Data are means TSE
(n= 9). HR was significantly greater than 15 and 30 min at 45 min
andbeyondinbothtrials(PG0.05). Tc was significantly greater than
15 min for all time points in both trials (PG0.05). RPE was signifi-
cantly greater than 15 min from 60 to 120 min in both trials (PG0.05).
*Significantly greater than HYD trial (PG0.05). Arrows (,) indicate
approximately 1% and 2% BM loss.
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significantly lower in the DEH (47 T1 g) versus HYD (56 T
1 g) trials.
Cardiovascular, thermoregulatory, and RPE re-
sponses. HR significantly increased over time in both tri-
als and became significantly greater than 15 and 30 min at
45 min and beyond in both trials. Subjects had a signifi-
cantly higher HR from 30 to 120 min of cycling in the DEH
versus HYD trial (Fig. 2A). Tc was significantly greater than
15 min for all time points in both trials. In the DEH trial,
Tc was significantly greater than the HYD trial from 30 to
120 min (Fig. 2B). RPE significantly increased over time in
both trials and was significantly greater than 15 min from 60
to 120 min. RPE was significantly greater in the DEH ver-
sus HYD trial from 60 to 120 min (Fig. 2C). The mean RPE
over the entire 120-min trial was significantly greater in the
DEH trial (DEH, 13.9 T0.6, vs. HYD, 12.3 T0.4).
Blood measurements. Blood Hb and Hct were sig-
nificantly higher than rest from 20 to 120 min of exercise in
both trials (Table 2). In the DEH trial, Hb was significantly
greater than the HYD trial from 40 to 120 min (Table 2).
There was a trend for Hct to be higher in the DEH trial
throughout the exercise, but the difference did not reach sig-
nificance until 120 min of exercise (Table 2). Pvol loss was
significantly greater than rest in both trials at all exercise
time points and significantly greater in the DEH versus
HYD trial from 40 to 120 min (Table 2). Blood glucose was
significantly lower than rest between 40 and 120 min in both
trials, with no difference between trials (Table 2). Blood lac-
tate was significantly increased from rest at 20 min of ex-
ercise and beyond in both trials and was also significantly
higher in the DEH versus HYD trial at all exercise time points
(Table 2). Plasma FFA and EPI significantly increased from
rest at all time points in both trials, with no significant dif-
ferences between trials (Table 2).
Muscle fuels and metabolites. Skeletal muscle PCr
significantly decreased in the first 60 min of exercise in both
trials and remained significantly lower than rest at 120 min
of exercise in both trials, but was not significantly different
between trials (Table 3). Cr increases were reciprocal with
the PCr decreases, and muscle ATP content was not signif-
icantly changed with exercise or between trials (Table 3).
Muscle free ADP and AMP significantly increased in the
first 60 min of exercise in both trials and remained signifi-
cantly higher than rest at 120 min of exercise in both trials,
but was not significantly different between trials (Table 3).
Muscle lactate content increased with exercise and peaked
at 60 min in both trials and was significantly greater at
60 min in the DEH versus HYD trial (Table 3). Muscle gly-
cogen content was similar in the two trials before exer-
cise and significantly lower at 60 and 120 min in both
TABLE 2. Whole Hb, Hct, glucose, lactate, Pvol loss, plasma FFA, EPI, and concentration during 120 min of cycling at approximately 65% V
˙O
2peak
in the hydrated (HYD) and dehydrated
(DEH) trials.
Time (min) Trial 0 20 40 60 80 100 120
Hb (gI100 mL)
j1
HYD 11.9 T0.4 12.9 T0.5 12.6 T0.3 12.8 T0.3 12.6 T0.3 12.7 T0.3 12.6 T0.3
DEH 11.8 T0.4 12.9 T0.5 13.3 T0.6* 13.3 T0.4* 13.4 T0.5* 13.2 T0.3* 13.3 T0.4*
Hct (%) HYD 40.3 T1 41.1 T0.8 41.2 T1 42.3 T1.5 42.2 T0.9 42.4 T0.8 42.0 T0.9
DEH 39.8 T1.1 41.6 T1.2 41.6 T1.1 43.0 T0.8 42.8 T0.9 42.8 T0.9 44.3 T0.7*
Pvol loss (%) HYD —– j3.6 T0.9 j4.6 T0.7 j6.2 T1.9 j5.5 T1.5 j5.8 T1.0 j5.3 T1.2
DEH —– j3.3 T1.4 j8.2 T0.9* j9.6 T1.1* j9.5 T0.9* j10.1 T1.1* j11.3 T1.2*
Glucose (18) HYD 4.5 T0.1 4.1 T0.2 4.0 T0.1 3.9 T0.2 3.8 T0.1 3.8 T0.1 4.0 T0.2
DEH 4.5 T0.1 4.2 T0.1 4.2 T0.1 4.1 T0.1 4.0 T0.1 4.1 T0.1 4.1 T0.1
Lactate (18) HYD 0.8 T0.1 1.5 T0.4 1.3 T0.7 1.3 T0.3 1.0 T0.2 1.4 T0.4 1.0 T0.4
DEH 0.8 T0.1 2.2 T0.6* 1.9 T0.5* 2.0 T0.6* 2.3 T0.7* 2.0 T0.6* 2.4 T0.7*
Plasma FFA (18) HYD 0.2 T0.04 —– —– 0.4 T0.1 0.6 T0.1 —– 1.0 T0.2
DEH 0.2 T0.04 —– —– 0.3 T0.1 0.4 T0.1 —– 0.7 T0.1
Plasma EPI (nM) HYD 0.8 T0.1 —– —– 1.3 T0.2 1.4 T0.2 —– 1.5 T0.3
DEH 0.7 T0.1 —– —– 1.2 T0.1 1.3 T0.1 —– 1.4 T0.2
Values are means TSE (n= 9).
* Significantly greater than HYD (PG0.05).
Blood Hb, Hct, Pvol loss, blood lactate, plasma FFA, and EPI were significantly greater than 0 min at all time points in both trials (PG0.05). Blood glucose was significantly lower than 0
min from 40 to 120 min in both trials (PG0.05).
TABLE 3. Skeletal muscle fuel and metabolite contents during 120 min of cycling at approximately 65% V
˙O
2peak
in the hydrated (HYD) and dehydrated (DEH) trials.
HYD DEH
Time (min) 0 60 120 0 60 120
PCr (mmolIkg
j1
dm) 80.1 T4.3 60.1 T6.2 55.7 T7.4 81.1 T4.1 59.3 T5.6 56.0 T6.9
Cr (mmolIkg
j1
dm) 81.0 T3.8 101.0 T5.2 105.3 T8.9 80.2 T3.8 102.0 T7.8 105.5 T6.7
ATP (mmolIkg
j1
dm) 24.9 T1.2 23.3 T1.0 24.4 T1.3 23.9 T1.4 24.1 T3.5 24.9 T1.7
ADPf (KmolIkg
j1
dm) 133.1 T11.8 230.5 T27.7 246.7 T49.4 134.0 T5.5 231.0 T32.1 279.9 T43.1
AMPf (KmolIkg
j1
dm) 0.7 T0.1 2.5 T0.7 3.4 T1.3 0.7 T0.1 2.3 T0.5 3.5 T0.9
Lactate (mmolIkg
j1
dm) 3.3 T0.3 6.1 T1.1 7.1 T1.2 3.3 T0.6 10.2 T2.3* 8.4 T1.7
Glycogen (mmolIkg
j1
dm) 425 T36 223 T41 173 T34 461 T28 223 T26 131 T18
Values are means TSE (n= 9).
* Significantly higher than HYD trial (PG0.05).
PCr and glycogen were significantly lower than 0 at 60 and 120 min. Cr, ADPf, AMPf, and lactate were significantly greater than 0 at 60 and 120 min.
EFFECTS OF DEHYDRATION ON MUSCLE METABOLISM Medicine & Science in Sports & Exercise
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trials compared with rest (Table 3). There was no signifi-
cant difference in glycogen use (17%) in the first 60 min
of exercise between trials (DEH, 238 T38, vs. HYD,
203 T52 mmolIkg
j1
dry muscle (dm)). There was signifi-
cantly more glycogen used (84%) from 60 to 120 min of
exercise in the DEH (92 T13 mmolIkg
j1
dm) versus
HYD (50 T14 mmolIkg
j1
dm) trials and significantly more
glycogen used (31%) during the entire DEH (330 T
33 mmolIkg
j1
dm) versus HYD (252 T49 mmolIkg
j1
dm)
trials (Fig. 3).
DISCUSSION
This study investigated the effects of mild progressive de-
hydration during exercise at approximately 65% V
˙O
2peak
on
whole body substrate oxidation and skeletal muscle metab-
olism, as well as cardiovascular, thermal, and mental re-
sponses in recreationally active, hydrated females. In the
control trial (HYD) of this study, we maintained hydration
by having subjects drink enough fluid to precisely replace
their sweat losses over the 120-min cycling trial. During this
trial, HR increased from 150 T5 bpm at 20 min to 158 T5
and 165 T5 bpm at 60 and 120 min, whereas Tc increased
from 37.3-CT0.2-C at rest to 37.9-CT0.1-C at 15 min and
38.2-CT0.1-C and 38.5-CT0.2-C at 60 and 120 min. In
the DEH trial, when the subjects progressively dehydrated
by sweating, they lost approximately 1% and 2% BM at 60
and 120 min and added progressive dehydration to the phys-
iological demands of exercising for 120 min at approxi-
mately 65% V
˙O
2max
. All physiological responses to exercise
were exacerbated in the DEH trial as HR increased from
154 T5 at 20 min to 163 T5, and 176 T5 bpm at 60 and
120 min, representing increases of 4–9 bpm at these time
points. Subjects also had elevated Tc values (37.3-CT0.2-C
at rest and 38.1-CT0.2-C, 38.7-CT0.2-C, and 39.1-CT
0.2-C at 20, 60, and 120 min) and were 0.2-C, 0.5-C, and
0.6-C higher at 20, 60 and 120 min in the DEH versus HYD
trial. Even in the first hour of exercise in DEH (approxi-
mately 1% BM loss), RPE, Pvol loss, and blood (La) were
all higher, and there was a significantly greater reliance on
whole body carbohydrate, higher muscle lactate content, and
a trend for higher muscle glycogen use (P= 0.15). In the
second hour, BM loss progressed from 1% to 2%, and the
additional physiological parameters remained higher, and
whole body carbohydrate oxidation and muscle glycogen
use were also significantly greater in the DEH trial. The 2%
BM loss for 2 h of exercise increased whole body carbo-
hydrate oxidation by 9% and muscle glycogen use by 31%
in female subjects who were hydrated before exercise.
The effects of dehydration on substrate oxidation
and muscle metabolism. Hargreaves et al. (17) demon-
strated in trained males that a 3% BM loss over a 2-h trial
resulted in a significantly higher whole body RER after 60
and 120 min of exercise compared with the euhydrated trial.
They also observed a 16% greater muscle glycogen use over
the entire trial with fluid restriction. Similar results were
reported by Gonzalez-Alonso et al. (15) who had male sub-
jects cycle until volitional exhaustion (135 T4 min) in the
heat (35-C) while progressively dehydrating to approxi-
mately 3.9% BM loss. They reported increased carbohydrate
oxidation, muscle glycogen use (45% greater), muscle lac-
tate accumulation, and net lactate release across the con-
tracting leg compared with the euhydrated trial. In contrast,
Walsh et al. (37) demonstrated that mild dehydration of
1.3% BM loss in trained males did not change RER after
60 min of cycling at 70% V
˙O
2peak
in the heat (32-C). It
seems likely that the low level of dehydration may be the
reason for no effect on RER. In comparison, the present
study with a neutral environment, which was conducted on
recreational trained females, demonstrated that RER was sig-
nificantly higher in the DEH trial as early as 40 min of cy-
cling when dehydration was G1% BM loss and remained
significantly higher than the HYD trial for the duration of
the trial. The present RER and glycogenolysis data matches
Hargreaves et al. (17) despite this study being conducted on
recreationally trained females and not trained males. As
well, our results suggest that a portion of the increased py-
ruvate production in the DEH trial was oxidized and some
was converted to lactate.
The major question is, ‘‘What accounts for the increased
glycogenolysis in the DEH trial?’’ There are three main
hypotheses that have been proposed to explain the substrate
shift toward greater carbohydrate metabolism and muscle
glycogenolysis during exercise and heat stress; 1) an aug-
mented sympathoadrenal response leading to greater glyco-
gen phosphorylase (PHOS) activation and flux, 2) increased
allosteric activation of glycogen PHOS via increased free ADP
and AMP (energy status of the cell) levels, and 3) higher in-
tramuscular temperature during exercise when dehydrated
(8). Hargreaves et al. (17) reported no difference in plasma
EPI at 60 or 120 min of cycling with 3% BM loss but signif-
icantly greater plasma norepinephrine content only at 120 min.
The authors suggested that fluid ingestion during exercise
FIGURE 3—Muscle glycogen use during 120 min of cycling at approx-
imately 65% V
˙O
2peak
in the hydrated (HYD) and dehydrated (DEH)
trials. Data are means TSE (n= 9). *Significantly greater than HYD trial
(PG0.05).
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attenuates the normal exercise-induced increase in EPI, and
the blunting of the sympathoadrenal activity may be due to
hydration status and Tc, but it is difficult to assess these
factors independently. The present study also found no dif-
ference in EPI response with fluid restriction (2% BM loss)
in females, which downplays the role of EPI as the reason
for the significantly greater muscle glycogen use and mus-
cle lactate content at 60 min while exercising dehydrated.
The energy status of the cell (free ADP and AMP) exerts
powerful allosteric regulation of glycogen PHOS and there-
fore plays a vital role in determining the rate of glycogenoly-
sis. In light of the augmented glycogen use accompanying
progressive DEH, we predicted that the energy status of the
cell may be decreased (higher free ADP and AMP) to agreater
extent when dehydrated and explain the accelerated glycogen
use. However, this was not observed, suggesting that the en-
ergy status of the cell was not altered by mild dehydration.
Previous work suggests that higher Tc and Tm are respon-
sible for the increased glycogenolysis and increased reli-
ance on carbohydrate oxidation for muscle ATP production
(9–11,15,34). Research investigating local hyperthermia in
the working muscle demonstrated an increase in Tm, mus-
cle glycogenolysis, and muscle (La), independent of changes
in circulating EPI (17,32). Starkie et al. (34) cooled one leg
before two-legged cycling and reported increased glyco-
genolysis in noncooled or hotter leg when both legs were
exposed to the same (EPI). Febbraio (9) concluded in a re-
view that increases in Tc of 90.5-C significantly increased
intramuscular carbohydrate use during moderate intensity
exercise in the heat. In the present study, in a neutral envi-
ronment, Tc was already 0.2-C–0.5-C higher from 20 to
60 min of exercise in the DEH trial. Although Tm was not
measured in this study, the work from Hargreaves’ labora-
tory (7,9,34) suggests that Tm would also have been higher
in the first hour of exercise of the present study. Therefore,
Tm (Q10 effect) appears to be the primary mechanism in-
ducing the shift in intramuscular glycogenolysis and whole
body carbohydrate oxidation and during progressive dehy-
dration in females. It is currently unknown why dehydration
preferentially increases CHO metabolism and not fat me-
tabolism and future research needs to further elucidate the
effect of dehydration on the perturbations to intramuscular
metabolism. One would predict that the Q10 effect would
also increase fat metabolism; however, the results of this
study demonstrate that fat oxidation was reduced with mild
dehydration, and carbohydrate oxidation was more sensitive
than fat oxidation to increases in Tc. The down-regulation
of fat oxidation cannot be explained by FFA delivery be-
cause there was no difference between trials in plasma FFA,
nor can the effect of muscle pH on carnitine palmitoyl
transferase 1 be considered as a mechanism for the de-
creased fat oxidation because there was no significant dif-
ference in muscle acidity between trials. This has been
reported in the past because Montain et al. (23) had subjects
perform knee-kicking exercise to exhaustion (approximately
250 s) when hypohydrated to 4% BM or euhydrated. Al-
though performance was reduced by 15% in the hypohy-
drated trial (213 vs. 251 s), they reported no change in
muscle pH or ATP levels between trials. Moreover, mild
dehydration may affect the uptake of FFA into the muscle or
alter intramuscular triacylglycerol breakdown; however, this
is merely speculation, and the mechanisms by which dehy-
dration causes a reduction in fat oxidation remain unclear
and call for further investigation.
Effects of dehydration on cardiovascular and ther-
mal responses. It is well established that fluid ingestion
attenuates the increases in HR and Tc and the decreases in
stroke volume and cardiac output that occur during prolonged
exercise without fluid ingestion (1,2,6,11,16,19,20,32). An
early study demonstrated that when heat-acclimatized male
subjects were dehydrated to 3%, 5%, and 7% BM loss by an
exercise-heat regime and then walked in a hot environment
(49-C) at a low intensity for 140 min, HR and Tc increased
linearly with the severity of dehydration (32). In a similar
way, our results demonstrated that as dehydration increased
from 0% to 1% and 1% to 2% BM during exercise in the DEH
trial, HR and Tc became progressively higher than the ele-
vations in the HYD trial. Hypovolemia and the displacement
of blood to the skin for evaporative cooling make it difficult
to maintain central venous pressure (CVP) during exercise
when fluid is restricted (31). CVP is regulated by the contin-
uous adjustment of blood volume to the changing size of
the vascular bed to maintain cardiac output, and heat stress
and/or exercise-induced dehydration provides a threat to this
control because inadequate fluid intake during periods of
sweat loss reduces Pvol (24). In light of the significantly
greater loss in Pvol found in the DEH versus the HYD trial
after approximately 20 min of cycling, a reduction in CVP
and stroke volume may account for the significantly elevated
HR to maintain cardiac output when stroke volume was com-
promised. An accompanying baroreflex that would decrease
cutaneous blood flow and heat transfer to the periphery lead-
ing to heat storage may account for the augmented Tc found
in the DEH trial. In support of this, Nadel et al. (25) reported
that diuretic-induced dehydration of 2.7% BM loss led to
restrictions in core-to-skin heat transfer, which forced esoph-
ageal temperature to nearly 39-C during 30 min of cycling at
55% V
˙O
2peak
in the heat compared with 38.4-Cineuhydrated
subjects. Montain and Coyle (22) investigated whether fluid
ingestion attenuated the hyperthermia and cardiovascular drift
that occurred during exercise dehydration due to increases in
blood volume. Seven trained male subjects exercised at ap-
proximately 65% V
˙O
2peak
for 2 h in three conditions; no fluid
replacement, infusion with a blood volume expander, or given
fluid to replace approximately 80% of sweat loss. They re-
ported that fluid replacement and the blood volume expander
treatment maintained blood volume compared with the no
fluid trial, but only fluid replacement resulted in lower Tc. The
authors argued that the decreased hyperthermia during exer-
cise in the fluid replacement trial was due to the measured
increase in skin blood flow. In the present study, female sub-
jects had higher Tc values in the last 90 min of exercise in the
EFFECTS OF DEHYDRATION ON MUSCLE METABOLISM Medicine & Science in Sports & Exercise
d
1955
APPLIED SCIENCES
Copyright © 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
DEH versus HYD trials, whereas the sweat rates were simi-
lar, suggesting that the lack of heat transfer to the periphery
accounted for the elevated Tc in the DEH trial with as little
as 1%–2% BM loss.
Effects of dehydration on ratings of perceived
exertion. In this study, RPE mirrored the rise in HR and
Tc with progressive dehydration and became significantly
higher in the DEH trial at 60 min of cycling when subjects
had lost approximately 1% BM. Similar results have been
reported in other studies investigating the effects of progres-
sive dehydration on RPE (16,20). It is speculated that hy-
povolemia associated with exercise dehydration leading to
a reduction in brain blood flow may exasperate the displea-
sure associated with exercising without fluid leading to
greater perceived exertion (18). More simply, it may be that
the elevations in Tc, HR, and reduced Pvol in the DEH trial
are sensed, and the feedback to the brain results in the
greater RPE during exercise at the same relative intensity in
a mildly dehydrated state. Shirreffs et al. (33) reported that
as subjects became progressively more dehydrated to 2.7%
BM loss, they reported feelings of headache and reductions
in their ability to concentrate and their alertness was re-
duced, which are all contributing factors to an elevated RPE
during exercise.
CONCLUSIONS
This study is the first to investigate the time course of
changes in whole body substrate oxidation and skeletal mus-
cle metabolism, as well as cardiovascular, thermal, and men-
tal responses in recreationally active, hydrated females
with progressive mild dehydration during exercise at
approximately 65% V
˙O
2peak
in a neutral environment.
Moreover, total carbohydrate oxidation and muscle glyco-
genolysis were significantly increased early in exercise
when BM loss was G1% to 2%, which we attribute to de-
hydration-induced increases in Tc and skeletal Tm, because
there were no differences in plasma EPI or the energy
status of the cell (free ADP or AMP) between the HYD and
DEH trials. In addition, the traditional changes in physio-
logical parameters accompanying exercise in a HYD
state were exacerbated with mild dehydration of 1%–2%
BM loss.
This research was funded by the Gatorade Sports Science Insti-
tute. No other funding was received.
Each authors report no conflict of interest.
The results of this study do not constitute endorsement by the
American College of Sports Medicine.
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