J Appl Physiol 103: 1242–1250, 2007.
First published July 19, 2007; doi:10.1152/japplphysiol.00560.2007.
Single muscle fiber function with concurrent exercise or nutrition
countermeasures during 60 days of bed rest in women
Scott Trappe, Andrew Creer, Dustin Slivka, Kiril Minchev, and Todd Trappe
Human Performance Laboratory, Ball State University, Muncie, Indiana
Submitted 23 May 2007; accepted in final form 16 July 2007
Trappe S, Creer A, Slivka D, Minchev K, Trappe T. Single
muscle fiber function with concurrent exercise or nutrition coun-
termeasures during 60 days of bed rest in women. J Appl Phys-
iol 103: 1242–1250, 2007. First published July 19, 2007;
doi:10.1152/japplphysiol.00560.2007.—There is limited information
on skeletal muscle properties in women with unloading and counter-
measure programs to protect the unloading-induced atrophy. The
current investigation tested the hypothesis that a concurrent aerobic
and resistance exercise training program would preserve size and
contractile function of slow- and fast-twitch muscle fibers. A second-
ary objective was to test the hypothesis that a leucine-enriched
high-protein diet would partially attenuate single fiber characteristics.
Vastus lateralis muscle biopsies were obtained before and on day 59
of bed rest from a control (BR; n ⫽ 8), nutrition (BRN; n ⫽ 8), or
exercise (BRE; n ⫽ 8) group. Single muscle fibers were studied for
diameter, peak force (Po), contractile velocity, and power. Those in
the BR group had a decrease (P ⬍ 0.05) in myosin heavy chain
(MHC) I diameter (⫺14%), Po (⫺35%), and power (⫺42%) and
MHC IIa diameter (⫺16%) and Po (⫺31%; P ⫽ 0.06) and an increase
(P ⬍ 0.05) in MHC hybrid fibers. Changes in size and function of
MHC I (⫺19 to ⫺44%) and IIa (⫺21% to ⫺30%) fibers and MHC
distribution in BRN individuals were similar to results in the BR
group. In BRE conditions, MHC I and IIa size and contractile function
were preserved during bed rest. These data show that the concurrent
exercise program preserved the myocellular profile of the vastus
lateralis muscle during 60-day bed rest. To combat muscle atrophy
and function with long-term unloading, the exercise prescription
program used in this study should be considered as a viable training
program for the upper leg muscles, whereas the nutritional interven-
tion used cannot be recommended as a countermeasure for skeletal
muscle.
skeletal muscle; contractile properties; microgravity; spaceflight;
WISE-2005 study
UNLOADING-INDUCED ATROPHY of skeletal muscle has been well
documented (1). However, the overwhelming majority of this
information has been based on studies conducted in men, with
little information available concerning the effects of unloading
on skeletal muscle function in women (26). Our review of the
skeletal muscle literature indicates that only 7% (n ⫽ 35) of the
⬃500 bed rest subjects in the last 40 years have been women,
with no studies focusing exclusively on women. Because
women will certainly be involved in future spaceflight mis-
sions, more information is needed in this population to better
understand how skeletal muscle responds to prolonged unload-
ing and exercise. Furthermore, information on muscle atrophy
in women would be applicable to the general health care industry
for various clinical conditions (i.e., cachexia, sarcopenia).
Address for reprint requests and other correspondence: S. Trappe, Human
Performance Laboratory, Ball State Univ., Muncie, IN 47306 (e-mail:
strappe@bsu.edu).
1242 8750-7587/07 $8.00 Copyright © 2007 the American Physiological Society
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Developing specific countermeasures to negate the unload-
ing-induced atrophy at the whole muscle and cellular levels is
warranted to promote long-term space exploration and for its
potential use in clinical situations. A recent 90-day bed rest
study involving male volunteers tested the efficacy of a resis-
tance-training countermeasure to combat reduced skeletal mus-
cle function and mass that occur during unloading (3). Al-
though whole muscle strength and mass were maintained by
resistance exercise during bed rest, cellular data from the
vastus lateralis of resistance-trained subjects showed an incom-
plete preservation of slow-twitch [myosin heavy chain (MHC) I]
muscle fiber size and function (46). In addition, a slow-to-fast
shift in MHC isoform composition with a concomitant increase
in fibers expressing multiple MHC isoforms (i.e., hybrid fibers)
was also observed (21). These findings highlight that resistance
exercise alone was insufficient to maintain slow-twitch fiber
structure and function during long-duration bed rest in men. As
a result, a more comprehensive exercise countermeasures pro-
gram needs to be identified that effectively targets and protects
both slow- and fast-twitch muscle fibers in human skeletal
muscle. This information will be important to ensure the safety
and work capacity of humans engaged in long-duration space
travel and planetary exploration.
One possible solution would be the integration of aerobic
exercise into the resistance training program, since aerobic
training mainly targets slow-twitch muscle fibers (41) and has
historically been part of the on-orbit exercise countermeasure
program. Studies investigating the effects of concurrent resis-
tance and endurance exercise have reported an inhibitory effect
on strength and hypertrophy compared with resistance training
alone (4, 15, 27). Although potential interference between
resistance and endurance training may prevent optimal muscle
strength and mass gains, a concurrent training program does
improve whole muscle strength and size (4, 15, 27). At the
single fiber level, concurrent training has been shown to
preserve or increase slow- and fast-twitch muscle fiber size (4,
25, 38), as well as decrease hybrid muscle fiber types (38). To
the best of our knowledge, concurrent training protocols as a
countermeasure for skeletal muscle during prolonged unload-
ing have not been investigated. The findings derived from
ground-based training studies suggest that the combination of
a resistance and aerobic exercise training regimen may provide the
balanced stimulus necessary to fully protect unloaded human
skeletal muscle by targeting both MHC I and IIa fibers and
attenuating a slow-to-fast shift in MHC isoform composition.
The anabolic effect of amino acids (39, 51) has recently
received attention as a potential countermeasure for skeletal mus-
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.
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 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE
cle during periods of unloading and would be advantageous in
situations where exercise may not be possible. Previous studies
have shown that amino acids stimulate muscle protein synthesis
(8, 9, 12, 34). Independently, the amino acid leucine has been
shown to have strong anabolic (10, 29, 30, 40) and anti-catabolic
(10, 13, 20, 36, 44) effects on skeletal muscle protein kinetics.
Shorter duration bed rest studies support the idea for a nutritional
countermeasure to promote the preservation of muscle mass
during unloading (7, 37, 42, 43). In a recent 28-day bed rest study,
a nutritional countermeasure consisting of additional amino acids
resulted in conflicting results showing that whole body lean
muscle mass was maintained (37) but not vastus lateralis MHC I
and IIa fiber size (19). To date, the efficacy of an amino acid
countermeasure has not been tested in longer duration bed rest or
in women during periods of unloading.
The primary goal of this investigation was to test the
hypothesis that a concurrent training protocol consisting of
resistance and aerobic exercise would preserve vastus lateralis
MHC I and IIa single muscle fiber structure and contractile
function. A secondary objective of this investigation was to test
the hypothesis that a leucine-enriched high-protein diet would
partially preserve vastus lateralis MHC I and IIa muscle fiber
size and contractile function. A more complete report of the
whole muscle performance and mass are presented by our
research team elsewhere (48).
METHODS
Overall Study Design
This investigation integrated several physiological, metabolic, and
psychological experiments to examine the responses to prolonged bed
rest (i.e., simulated weightlessness) in women. The primary goal of
this study was to test specific exercise and nutritional countermeasure
programs to the expected deleterious effects of long-term bed rest.
The study was conducted at the Institute for Space Physiology and
Medicine (MEDES) in Toulouse, France.
The study design consisted of 20 days of baseline data collection
(BDC), 60 days of 6° head-down tilt (HDT) bed rest, and a recovery
period that varied in duration depending on the specific measure-
ments. The BDC period provided time for the subjects to equilibrate
to the standardized diet during BDC for each of the volunteers. All
meals and snacks were prepared for the volunteers. Body weight was
measured daily (during bed rest in the 6° HDT position). During bed
rest, the volunteers remained in the 6° HDT position or horizontal
position for all activities, including eating, bathing, excretory func-
tion, and physiological testing and training (0° for treadmill). Contin-
uous video and medical staff monitoring ensured compliance to the
bed rest period.
The study was completed in two separate campaigns, with each
campaign consisting of 12 subjects, four from each experimental
group: bed rest only (BR), bed rest with the exercise countermeasure
(BRE), and bed rest with the nutritional countermeasure (BRN). The
BDC, bed rest, and initial recovery periods of both campaigns were
completed in one calendar year (2005). Because similar changes
were observed in campaign 1 and campaign 2, the data were com-
bined for each group.
Subjects
Twenty-four healthy women from the European Union were re-
cruited and underwent 60 days of 6° HDT bed rest. Subjects were
divided into bed rest only (n ⫽ 8), bed rest ⫹ nutritional counter-
measure (n ⫽ 8), or bed rest ⫹ exercise countermeasure (n ⫽ 8)
groups. Subject characteristics are shown in Table 1. Screening for
potential volunteers took place at the MEDES clinic and involved an
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1243
Table 1. Subject characteristics
Group Age, yr Height, cm Study Period Weight, kg
BR 34⫾1 163⫾2 Pre 55.6⫾1.4
Bed rest 53.4⫾1.4
HDT60 52.3⫾1.3 (⫺3.3⫾0.2)
Post 53.0⫾1.4
BRN 29⫾1 170⫾2 Pre 61.2⫾1.6
Bed rest 59.4⫾1.4
HDT60 58.5⫾1.5 (⫺2.7⫾0.3)
Post 59.5⫾1.5
BRE 33⫾1 165⫾3 Pre 58.1⫾2.2
Bed rest 56.2⫾2.1
HDT60 55.2⫾2.2 (⫺2.9⫾0.2)
Post 55.2⫾2.2
Values are means ⫾ SE. Numbers in parentheses represent the absolute
change in body weight from pre-bed rest to HDT60. BR, bed rest only group;
BRN, bed rest ⫹ nutritional countermeasure group; BRE, bed rest ⫹ exercise
countermeasure group. Pre period consisted of the baseline data collection
(BDC) from ⫺10 to ⫺1 (the 10 days before the bed rest period). Bed rest
period consisted of 1 (HDT1) to 60 (HDT60) days of 6° head-down tilt. Post
period was a recovery period from R ⫹ 0 to R ⫹ 10 [recovery day 0 to recovery
day 10 (the post-bed rest phase)].
interview, general medical examination, and psychological evalua-
tion. Subjects were selected into each group by the MEDES staff
based on their medical and psychological profiles. Subjects were
recreationally active (pre-bed rest maximal oxygen consumption of
⬃39 ⫾ 1 ml 䡠 kg⫺1 䡠 min⫺1 for all groups) and excluded from partic-
ipation if they were sedentary or too involved with physical training.
Before the screening, each potential volunteer was informed of all
procedures and potential risks associated with the experimental test-
ing. Informed consent was then obtained from each volunteer. This
study was approved by the Human Use Committees in France and the
United States (Johnson Space Center and Ball State University).
Countermeasures to Bed Rest
Resistance training protocol. The BRE group trained the thigh
muscle groups using supine squat (SS) exercises on an inertial
ergometer (2, 3, 5). Resistance exercise was scheduled for each
subject approximately every third day (2–3 days/wk) beginning on
day 2 of bed rest for a total of 19 sessions. The inertial ergometer was
in the 6° HDT position, and all resistance exercises were performed in
the supine position. Ten minutes of light supine cycling and submaxi-
mal SS repetitions were completed as warm-up. The SS exercise
consisted of four sets of seven maximal concentric and eccentric
repetitions. There were 2 min of rest between sets. Force and flywheel
rotational velocity were measured with work and power calculated
throughout each repetition (2, 3, 5). This exercise protocol was similar
to a previous 90-day study conducted in men (3, 46).
Because of various medical aspects that arose during the bed rest
period, including the combined aerobic and resistance exercise counter-
measure program (soreness and injury) and occasional mild illness, not
all sessions were completed as planned. To help minimize the chance for
injury, the second bed rest campaign (half the subjects) had a ramp-up
phase for the resistance exercise sessions. The first three sessions were at
70, 80, and 90% of maximal effort, with all remaining sessions planned
as maximal effort. Data compiled at the end of both bed rest campaigns
provided a profile of the exercise sessions. For the first campaign, 76%
were conducted as planned, 16% were reduced effort, and 8% were
missed. For the second campaign, 88% were conducted as planned, 9%
were reduced effort, and 3% were missed. The submaximal and missed
resistance exercise sessions varied among the volunteers and were scat-
tered throughout the 60-day bed rest period.
Aerobic training protocol. The lower body negative pressure
(LBNP) treadmill device used for this study was similar to that
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1244 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE

described previously (11, 32, 49). Two to four days per week, exercise
subjects performed 40 min of exercise ranging from 40% to 80% of
pre-bed rest peak oxygen consumption, followed by 10 min of resting
LBNP (32, 49).
During the course of the 60-day bed rest, 29 exercise sessions were
prescribed for each BRE subject. Not all exercise sessions were
completed by all subjects due to illness, joint pain, and soreness from
prior exercise. One subject was unable to complete the first two
exercise sessions due to medical reasons but subsequently completed
all other sessions. In one subject, three exercise sessions were not
completed because of back or hip pain. One exercise session in each
of two subjects was canceled due to short-term illness (fever, upper
respiratory symptoms).
All BRE subjects completed their exercise at an LBNP level that
produced approximately one body weight for half of the bed rest
period. Thereafter, as their tolerance to exercise increased, all but one
of the eight subjects exercised at ⬎1.05 times body weight. In the
subject who was prone to presyncopal symptomatology, the average
exercise and post-exercise LBNP were reduced to ⬃90% body weight
for part of the countermeasure period. Of the exercise sessions
performed, the mean exercise time was 50 ⫾ 2 min. Across all
exercise sessions completed, the average LBNP was 52 ⫾ 3 mmHg,
which corresponded to a mean loading of 1.0 ⫾ 0.1 body wt.
Nutritional countermeasure. All meals were prepared for all three
groups by the MEDES dietary staff, with controlled amounts of total
energy and macronutrients (carbohydrate, fat, and protein) (Table 2)
as well as sodium, potassium, calcium, and fluid intake. The goal of
the nutrition countermeasure was to provide an additional amount of
protein and free leucine during the bed rest period for the BRN group.
All three groups received similar diets during the pre-bed rest period,
with the protein composition maintained at ⬃1.0 g 䡠 kg body
wt⫺1 䡠 day⫺1. The BR and BRE groups continued to receive this
amount of protein during the bed rest period, whereas the BRN group
received ⬃1.45 g 䡠 kg body wt⫺1 䡠 day⫺1. In addition, the BRN group
received 3.6 g/day of free leucine, 1.8 g/day of free valine, and 1.8
g/day of free isoleucine equally divided out over the three meals of the
day. Thus the total protein intake for the BRN group was ⬃1.6 g/kg
body wt/day. To compensate for the additional increase in energy
intake from protein in the BRN group, carbohydrate content was
reduced during the bed rest period. Energy intake for all three groups
was adjusted downward during bed rest due to the reduced energy
expenditure compared with the pre-bed rest period. Also during bed
rest, the BRE group received an additional amount of energy intake
equal to the energy expended during the exercise training sessions.
Muscle Biopsy
A muscle biopsy (6) was obtained from the vastus lateralis of each
subject before bed rest and on day 59 of bed rest (range 16 –24 h after
the last exercise session). The muscle biopsy was performed on day 59
to avoid interfering with other testing procedures being performed on
the final day of bed rest before subject reambulation.
A portion of the sample was sectioned into several longitudinal
pieces, placed in cold skinning solution (see below), and stored
at ⫺20°C for later analysis of single muscle fiber physiology. After a
single muscle fiber experiment, each single fiber was analyzed for
MHC composition as described below. Muscle samples were trans-
ported to the Human Performance Laboratory at Ball State University
where the single muscle fiber physiology experiments were completed
within a 4-wk period after the muscle biopsy.
Skinning, Relaxing, and Activating Solutions
The skinning solution contained (in mM) 125 potassium propi-
onate, 2.0 EGTA, 4.0 ATP, 1.0 MgCl2, 20.0 imidazole (pH 7.0), and
50% (vol/vol) glycerol. The compositions of the relaxing and activat-
ing solutions were calculated with the use of an interactive computer
program described by Fabiato and Fabiato (17). These solutions were
adjusted for temperature, pH, and ionic strength using stability con-
stants in the calculations (24). Each solution contained (in mM) 7.0
EGTA, 20.0 imidazole, 14.5 creatine phosphate, 1.0 free Mg2⫹, and
4.0 free MgATP, KCl, and KOH to produce an ionic strength of 180
mM and a pH of 7.0. The relaxing and activating solutions had a free
Ca2⫹ concentration of pCa 9.0 and pCa 4.5, respectively (where
pCa ⫽ ⫺log Ca2⫹ concentration).
Single Muscle Fiber Physiology Experiments
For each experiment, a 2- to 3-mm muscle fiber segment was
isolated from a muscle bundle and transferred to an experimental
chamber filled with pCa 9.0 solution. Each end of the fiber was then
securely fastened between a force transducer (model 400A, Cam-
bridge Technology) and a direct current torque motor (model 308B,
Cambridge Technology) as described by Moss (35). The instrumen-
tation was arranged so that the muscle fiber could be rapidly trans-
ferred back and forth between experimental chambers filled with
relaxing or activating solutions. The apparatus was mounted on a
microscope (Olympus BH-2) so that the fiber could be viewed (⫻800)
during an experiment. Using an eyepiece micrometer, sarcomeres
along the isolated muscle segment length were adjusted to 2.5 ␮m,
and the fiber length (FL) was determined. All single muscle fiber
experiments were performed at 15°C.
Unamplified force and length signals were sent to a digital oscillo-
scope (Nicolet 310, Madison, WI), enabling muscle fiber performance to
be monitored throughout data collection. Analog force and position
signals were amplified (dual differential amplifier, model 300-DIF2,
Positron Development, Inglewood, CA), converted to digital signals
(National Instruments), and transferred to a computer (Gateway, Irvine,

Table 2. Nutritional overview for the study

Group Study Period Energy Intake, kcal Carbohydrate, g Fat, g Protein, g

BR Pre 1856⫾26 268⫾3 (58%) 61⫾1 (30%) 58⫾2 (12%)

Bed rest 1557⫾21 219⫾3 (56%) 52⫾1 (30%) 54⫾1 (14%)
Post 1830⫾56 265⫾9 (58%) 61⫾2 (30%) 54⫾2 (12%)
BRN Pre 1918⫾29 271⫾4 (56%) 64⫾1 (30%) 65⫾2 (14%)
Bed rest 1651⫾29 202⫾4 (49%) 55⫾1 (30%) 86⫾2 (21%)
Post 1906⫾65 271⫾9 (57%) 65⫾2 (31%) 60⫾2 (13%)
BRE Pre 1861⫾55 265⫾7 (57%) 62⫾2 (30%) 61⫾2 (13%)
Bed rest 1758⫾70 250⫾11 (57%) 59⫾2 (30%) 56⫾3 (13%)
Post 1866⫾70 266⫾11 (57%) 64⫾2 (31%) 57⫾2 (12%)
Values are means ⫾ SE. Numbers in parentheses are percent contribution of each nutrient to the total energy intake. BRN group also received 3.6 g/day of
free leucine, 1.8 g/day of free valine, and 1.8 g/day of free isoleucine (all 3 equally divided and consumed with the 3 meals of the day) during the bed rest period
for a total protein intake during the bed rest period of ⬃1.6 g 䡠 kg body wt⫺1 䡠 day⫺1. All 3 groups received similar protein composition during the pre- and post-bed
rest period (⬃1.0 g/kg body wt/day), and the BR and BRE groups continued to receive this amount of protein during the bed rest period.

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 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE
CA) for analysis using customized software. Servo-motor arm and
isotonic force clamps were controlled with a computer-interfaced force-
position controller (model 300-FC1, Positron Development).
For each single muscle fiber experiment, a fiber with a compliance
(calculated as FL divided by y-intercept) ⬎10% and/or a decrease in
peak force (Po) of ⬎10% was discarded and not used for analysis. The
within-fiber test/retest results of a single muscle fiber in our laboratory
for the measurements of size, force-power relationships, Po, and
contractile velocity were ⬍1%. The coefficients of variation for the
force transducer and servo-mechanical lever mechanism during the
1-yr period in which we examined single muscle cell function from
the women, as part of this investigation, were ⬍1%.
Single Muscle Fiber Analysis
Individual muscle fibers were analyzed for diameter, Po, maximal
unloaded shortening velocity (Vmax) (25), and force-power character-
istics. Detailed descriptions and illustrations of these procedures have
been previously published by our laboratory (45, 47).
Single fiber diameter. A video camera (Sony CCD-IRIS, DXC-
107A) connected to the microscope and interfaced to a computer
allowed viewing on a computer monitor and storage of the digitized
images of the single muscle fibers. Fiber diameter was determined
from a captured computer image taken with the fiber briefly sus-
pended in air (⬍5 s). Fiber width (diameter) was determined at three
points along the segment length of the captured image using NIH
public domain software (Scion Image, release Beta 4.0.2, for Win-
dows). Fiber diameter was calculated from the mean width of these
measurements, with the assumption that the fiber forms a cylindrical
cross-section when suspended in air.
Single fiber Po. The outputs of the force and position transducers
were amplified and sent to a microcomputer via a Lab-PC⫹ 12-bit
data acquisition board (National Instruments). Resting force was
monitored, and then the fiber was maximally activated in pCa 4.5
solution. Active Po was determined in each fiber by computer sub-
traction of the baseline force from the Po in the pCa 4.5 solution.
Single fiber shortening velocity from the slack. Fiber shortening
velocity from the slack (Vo) was measured by the slack-test technique
as described by Edman (16). The fiber was fully activated in pCa 4.5
solution and then rapidly released to a shorter length, such that force
fell to baseline. The fiber shortened, taking up the slack, after which
force began to redevelop. The fiber was then placed in pCa 9.0
solution and returned to its original length. The duration of unloaded
shortening, or time between onset of slack and redevelopment of
force, was determined by computer analysis. Four different activation
and length steps (150, 200, 250, and 300 ␮m; each ⱕ15% of FL) were
used for each fiber, with the slack distance plotted as a function of the
duration of unloaded shortening. Fiber Vo (FL/s) was calculated by
dividing the slope of the fitted line by the fiber segment length, and the
data were normalized to a sarcomere length of 2.5 ␮m.
Single fiber power. Submaximal isotonic load clamps were per-
formed on each fiber for determination of force-velocity parameters
1245
and power. Each fiber segment was fully activated in a pCa 4.5
solution and then subjected to a series of three isotonic load steps.
This procedure was performed at various loads so that each fiber was
subjected to a total of 15–18 isotonic contractions.
For the resultant force-velocity relationships, load was expressed as
P/Po, where P is the force during load clamping and Po is the peak
isometric force developed before the submaximal load clamps. Force
and shortening velocity data points derived from the isotonic contrac-
tions were fit by the hyperbolic Hill equation (28). Only individual
experiments in which r2 was ⱖ0.98 were included for analysis.
Fiber peak power was calculated from the fitted force-velocity param-
eters (Po, Vmax, and a/Po, where a is a force constant and Vmax is the
y-intercept). Absolute power (␮N䡠FL⫺1 䡠s⫺1) was defined as the product
of force (␮N) and shortening velocity (FL/s). Normalized power (W/l)
was defined as the product of normalized force and shortening velocity.
MHC Determination
After single muscle fiber physiology experiments were completed,
each fiber was solubilized in 80 ␮l of 1% SDS sample buffer and
stored at ⫺20°C until assayed (50). Briefly, samples were run over-
night at 4°C on a Hoefer SE 600 gel electrophoresis unit (San
Francisco, CA) utilizing a 3.5% (wt/vol) acrylamide stacking gel with
a 5% separating gel. After electrophoresis, the gels were silver stained
as described by Giulian et al. (22). MHC isoforms were identified
according to migration rate.
Whole Muscle Strength and Size
To document muscle strength changes, isometric and dynamic
muscle tests were performed before and after bed rest in all three
groups. MRI was conducted to determine size of the thigh muscles
before and after bed rest in all three groups. Muscle strength and size
procedures have been described in detail previously (3, 48).
Statistical Analysis and Calculations
Changes in MHC I and IIa single fiber variables [diameter, Po,
normalized force (Po/cross-sectional area; CSA), Vo, Vmax, peak
power, and normalized peak power] from pre- to post-bed rest in BR,
BRN, and BRE groups were determined by two-way ANOVA with
repeated measures. Only MHC I and IIa fibers were analyzed statis-
tically because of the low number of other fiber types studied in the
vastus lateralis muscle. However, data from the fiber types with
relatively low yield are presented. Significance was set at P ⬍ 0.05.
Post hoc analyses were conducted with a Tukey’s test. All data are
presented as mean values ⫾ SE.
RESULTS
A total of 925 successful single muscle fibers from the
vastus lateralis muscle were used for size, physiological func-
tion, and MHC analysis in this investigation (Table 3), corre-

Table 3. Single muscle fiber MHC composition of vastus lateralis fibers studied for physiological experiments from BR,
BRN, and BRE groups before and after 60 days of bed rest
Group MHC I MHC I/IIa MHC IIa MHC IIa/IIx MHC I/IIa/IIx Total

BR
Pre 52 (33%) 7 (4%) 76 (48%) 20 (13%) 1 (1%) 157
Post 48 (34%) 1 (1%) 35 (25%) 51 (37%) 4 (3%) 140
BRN
Pre 111 (66%) 3 (2%) 53 (31%) 2 (1%) 0 (0%) 169
Post 94 (69%) 8 (6%) 18 (13%) 15 (11%) 1 (1%) 137
BRE
Pre 81 (50%) 7 (4%) 62 (38%) 12 (7%) 0 (0%) 163
Post 70 (44%) 3 (2%) 69 (43%) 17 (11%) 0 (0%) 159
MHC, myosin heavy chain. Pre, before 60 days of bed rest; Post, after 60 days of bed rest.

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1246 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE

Table 4. Single muscle fiber diameter, Po, and normalized force (Po/CSA) for vastus lateralis MHC types from BR, BRN,
and BRE groups before and after 60 days of bed rest
MHC I MHC I/IIa MHC IIa MHC IIa/IIx

Pre Post Pre Post Pre Post Pre Post

Diameter, ␮m
BR 74⫾4§ 64⫾3*‡ 71⫾3 67⫾0 76⫾3 64⫾3* 66⫾3 60⫾2
BRN 88⫾3 72⫾2*a 83⫾5 73⫾3 78⫾1 62⫾6* 59⫾1 63⫾4
BRE 82⫾3 81⫾3 80⫾6 66⫾3 75⫾5 76⫾4 65⫾4 68⫾3
Po, mN
BR 0.42⫾0.04§ 0.27⫾0.03*‡ 0.60⫾0.05 0.29⫾0.00 0.62⫾0.05 0.43⫾0.06†‡ 0.55⫾0.04 0.41⫾0.03
BRN 0.63⫾0.07 0.38⫾0.03*‡ 0.56⫾0.05 0.42⫾0.04 0.70⫾0.04 0.49⫾0.09† 0.41⫾0.05 0.46⫾0.04
BRE 0.61⫾0.04 0.55⫾0.04 0.71⫾0.10 0.46⫾0.06 0.69⫾0.08 0.72⫾0.07 0.58⫾0.07 0.59⫾0.05
Po/CSA, kN/m2
BR 99⫾4 83⫾6*‡ 147⫾1 82⫾0 136⫾3 127⫾10 158⫾6 144⫾10
BRN 102⫾7 95⫾5 104⫾5 106⫾16 147⫾8 155⫾9 147⫾14 149⫾11
BRE 116⫾9 106⫾6 137⫾2 130⫾4 160⫾13 156⫾6 169⫾5 166⫾7
Values are means ⫾ SE. Po, peak force; CSA, cross-sectional area. *P ⬍ 0.05 from Pre; †P ⱕ 0.08 from Pre; ‡P ⬍ 0.05 from BRE; §P ⬍ 0.05 from BRN;
a
P ⫽ 0.08 from BRE.

sponding to all the functional data presented. An additional 56
fibers (BR ⫽ 18, BRN ⫽ 21, and BRE ⫽ 17) pre-bed rest and
42 fibers (BR ⫽ 16, BRN ⫽ 21, and BRE ⫽ 5) post-bed rest
were excluded for technical reasons as outlined previously
(46). This corresponded to 10% pre-bed rest and 8% post-bed
rest, which is typical for our laboratory in young healthy adults.
Single Muscle Fiber MHC Composition
The number of hybrid fibers studied after bed rest increased
(P ⬍ 0.05) 23% in BR and 19% in BRN compared with the
number of pure fibers (fibers consisting of a single MHC
isoform) studied (Table 3). The number of hybrid fibers studied
in BRE was similar (⬃12%) before and after bed rest. Because
of the small number of MHC I/IIa/IIx (n ⫽ 6) and IIx (n ⫽ 2)
fibers examined throughout the study, single fiber data from
these fibers were excluded.
Single Muscle Fiber Diameter
Pre- and post-bed rest single muscle fiber diameters are
shown in Table 4. Before bed rest, results in the BR group
showed smaller (P ⬍ 0.05) MHC I fiber diameter than in the
BRN group. Bed rest reduced (P ⬍ 0.05) MHC I and IIa
diameters, as shown in BR (⫺14 and ⫺16%) and BRN (⫺19%
and ⫺21%) samples. However, in BRE samples after bed rest,
MHC I or IIa diameter was unchanged. Although BRN sam-
ples showed larger MHC I fibers before bed rest, correlation
analysis between initial fiber diameter and fiber atrophy (ab-
solute change) was not significant (r2 ⫽ 0.37; P ⬎ 0.05).
Single Muscle Fiber Po and Po/CSA
Po and Po/CSA are shown in Table 4. Before bed rest, BR
samples showed lower (P ⬍ 0.05) MHC I fiber Po than BRN
samples. Po was decreased (P ⬍ 0.05) 35% and 40% in MHC
I fibers from both BR and BRN groups, respectively. A similar
trend (P ⬍ 0.08) was observed for MHC IIa single fiber Po in
both BR (⫺31%) and BRN (⫺30%) samples. For the BRE
group, there were no changes in MHC I or IIa single fiber Po
as a result of bed rest. When corrected for fiber size, MHC I
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fibers from the BR group had a 16% decline (P ⬍ 0.05) in
Po/CSA after bed rest.
Single Muscle Fiber Shortening Velocity (Vo and Vmax)
Contractile velocity was assessed by both the slack-test (25)
and force-velocity (Vmax) procedures (see METHODS). These
measurements provided a measure of shortening velocity as
shown in Table 5. There were no differences in contractile
velocity from pre- to post-bed rest within the BR, BRN, and
BRE groups.
Single Muscle Fiber Power
Absolute peak power and power normalized for muscle cell
size are shown in Table 6. In the BR and BRN groups, absolute
MHC I peak power was reduced (P ⬍ 0.05) 42% and 44%,
respectively. When normalized to muscle cell size, however,
these differences were not statistically significant. In the BRE
group, MHC I single fiber power parameters were maintained.
In the BR, BRN, and BRE groups, MHC IIa absolute peak and
normalized power were unchanged after bed rest.
Whole Muscle Size and Function
Thigh muscle size was decreased (P ⬍ 0.05) by 21% in the
BR and by 24% in the BRN group after bed rest. Thigh muscle
power was reduced (P ⬍ 0.05) by 27% in the BR group and by
33% in the BRN group after bed rest. In the BRE group, thigh
muscle size and whole muscle power were maintained with
bed rest. Complete whole muscle results are presented
elsewhere (48).
DISCUSSION
To our knowledge, this is the first study devoted to female
responses with prolonged bed rest and the influence of specific
exercise and nutrition countermeasures. Women are presently
part of and will continue to be part of the human space
program; however, there are few data describing the physio-
logical responses to unloading. The main findings from this
103 • OCTOBER 2007 • www.jap.org
 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE 1247
Table 5. Single muscle Vo and force-velocity procedures for vastus lateralis MHC types from BR, BRN, and BRE groups
before and after 60 days of bed rest
MHC I MHC I/IIa MHC IIa MHC IIa/IIx

Pre Post Pre Post Pre Post Pre Post

Vo, FL/s
BR 0.82⫾0.07 0.72⫾0.08 2.10⫾0.07 1.60⫾0.00 2.73⫾0.12 2.49⫾0.11 3.43⫾0.31 3.20⫾0.25
BRN 1.05⫾0.05 1.00⫾0.07 2.02⫾0.26 1.64⫾0.21 3.19⫾0.10 3.35⫾0.07 4.71⫾0.24 4.06⫾0.30
BRE 1.04⫾0.04 1.05⫾0.06 2.42⫾0.30 1.88⫾0.18 3.38⫾0.15 3.42⫾0.14 4.76⫾0.17 4.44⫾0.23
Vmax, FL/s
BR 0.63⫾0.05 0.57⫾0.07 1.52⫾0.10 1.29⫾0.00 2.32⫾0.11 2.32⫾0.07 3.05⫾0.29 2.88⫾0.22
BRN 0.85⫾0.05 0.87⫾0.07 1.69⫾0.22 1.48⫾0.18 2.92⫾0.12 3.05⫾0.90 4.56⫾0.17 3.50⫾0.32
BRE 0.67⫾0.04 0.77⫾0.04 1.67⫾0.26 1.69⫾0.22 2.86⫾0.21 3.12⫾0.11 4.52⫾0.21 4.20⫾0.23
Values are means ⫾ SE. FL, fiber length; Vo, fiber shortening velocity from the slack; Vmax, maximal velocity.

study, which used a combined resistance and aerobic exercise
program during bed rest, were that the MHC I and IIa fiber size
and contractile function were preserved from the vastus late-
ralis muscle (Figs. 1 and 2). These data show that a properly
balanced exercise protocol consisting of resistance and aerobic
exercise protects the myocellular function of unloaded skeletal
muscle in the upper leg of humans. The dietary intervention
provided no benefit for MHC I and IIa single muscle fiber size
or contractile function after the 60-day bed rest period. These
data suggest that nutrition alone does not appear to be an
alternative to exercise for protecting thigh skeletal muscle
during periods of long duration unloading.
Exercise Countermeasure
To date, there have been no studies in a 1-g or microgravity
environment to examine single muscle fiber contractile func-
tion with a concurrent training program. Most likely, concur-
rent exercise programs will be necessary to protect the various
physiological systems (for example, cardiovascular, muscle,
bone) of humans during long-duration space travel. The
present 60-day bed rest protocol that combined resistance and
aerobic exercises as a countermeasure for unloaded skeletal
muscle shows that cell size and function are maintained to
pre-bed rest levels. This is particularly noteworthy given the
magnitude of atrophy (⬃15%) and decline in contractile func-
tion (⬎40% in power) observed at the cell level in the control
group. Moreover, the addition of aerobic exercise provided an
adequate stimulus to maintain slow-twitch fiber size and func-
tion, without diminishing the positive benefits to the fast-twitch
fibers observed in the previous bed rest study utilizing only
resistance exercise (46). This is an important concept to con-
sider because some studies investigating the effects of concur-
rent resistance and endurance exercise have shown an attenu-
ation of muscle strength gains and hypertrophy compared with
resistance training alone (4, 15, 27). Although no fiber hyper-
trophy was observed in the exercise group in the present
60-day bed rest study, the maintenance of fiber size suggests a
balance between the anabolic and catabolic processes regulat-
ing muscle size (23) was achieved with the concurrent exercise
program during bed rest.
As with the 90-day study utilizing resistance training in men
(3), the concurrent exercise protocol used in this investigation
was also effective in maintaining thigh muscle mass and power
(48). In the 90-day study, this was primarily achieved by
protecting fast-twitch muscle fibers and increasing the propor-
tion of hybrid fibers containing the MHC IIa and/or IIx protein
in the vastus lateralis. Although advantageous for whole mus-
cle power, this cellular profile would lend itself to a more
fatigable muscle and would favor glycolytic metabolism, nei-
ther of which would be beneficial for endurance-based activi-
ties. In the present investigation, the maintenance of whole
muscle thigh characteristics with the exercise regimen was
reflective of cellular size and function (strength, speed, and
power) being maintained in both slow- and fast-twitch muscle
fibers. This protection of muscle quantity and quality is ideal
for a host of reasons ranging from crewmembers performing

Table 6. Single muscle fiber peak power and peak power normalized to cell size for vastus lateralis MHC types from BR,
BRN, and BRE groups before and after 60 days of bed rest
MHC I MHC I/IIa MHC IIa MHC IIa/IIx

Pre Post Pre Post Pre Post Pre Post

Peak power, ␮N 䡠 FL⫺1 䡠 s⫺1

BR 5.1⫾0.9 3.0⫾0.3*† 22.8⫾4.9 4.8⫾0.0 30.9⫾2.3 22.3⫾3.2 47.1⫾7.0 28.3⫾1.7
BRN 9.7⫾1.1 5.5⫾0.4*† 26.6⫾4.4 12.3⫾3.5 44.0⫾3.3 33.4⫾6.2 45.6⫾11.0 39.0⫾4.1
BRE 9.5⫾0.8 9.3⫾0.8 29.5⫾4.6 14.9⫾1.0 51.0⫾6.8 55.3⫾5.4 74.2⫾9.2 63.4⫾5.0
Peak power normalized to cell size, W/l
BR 1.20⫾0.14 0.95⫾0.07 5.35⫾0.84 1.36⫾0.00 6.99⫾0.44 6.77⫾0.86 13.43⫾1.60 10.35⫾0.90
BRN 1.59⫾0.11 1.37⫾0.09 4.64⫾0.23 3.36⫾1.23 9.24⫾0.70 10.80⫾0.93 16.20⫾3.69 13.20⫾2.00
BRE 1.89⫾0.23 1.82⫾0.10 6.04⫾0.98 4.62⫾0.62 12.17⫾1.72 12.10⫾0.62 21.71⫾1.00 18.17⫾1.25
Values are means ⫾ SE. *P ⬍ 0.05 from Pre; †P ⬍ 0.05 from BRE.

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1248 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE
Fig. 1. Summary of the vastus lateralis myosin heavy chain (MHC) I single
fiber parameters for subjects in bed rest only (BR), bed rest ⫹ nutrition (BRN),
or bed rest ⫹ exercise (BRE) groups before and after 60 days of bed rest. For
each variable, [diameter, peak force (Po; strength), maximal velocity (Vmax;
speed), and power], the percent change is shown.
extravehicular activities to the metabolic health of the individ-
ual given the key role that muscle has in a variety of physio-
logical processes.
We have previously shown that a subset of fibers used for
contractile physiology measurements (⬃15–20) is comparable
to a more detailed analysis (⬎100 fibers) after long-term bed
rest (21, 46), thus allowing for insight to MHC alterations from
the present data set. The concurrent training protocol prevented
an increase in the number of hybrid muscle fibers during bed
rest and did not alter the proportion of pure fiber types
(MHC I and IIa), as has been previously reported (46). A
limited number (n ⫽ 5 fibers) of hybrid fibers expressing MHC
I/IIa/IIx were observed in the present 60-day study after bed
rest. This MHC isoform is typically rare; however, the number
of fibers expressing MHC I/IIa/IIx increased ⬃16% in re-
sponse to 90-day bed rest (21, 46). The relatively high propor-
tion of hybrid fibers that we have observed in two long-
duration (60- and 90-day) bed rest studies is on the upper end
of what has been previously reported in humans. In the skeletal
muscle from inactive aging individuals (70 – 80 yr old), there
are ⬃35% hybrid fibers (14, 50). Only in extreme muscle
unloading perturbations, such as spinal cord injuries, are more
hybrid fibers and MHC IIx proteins present (33) than with
long-duration bed rest. This highlights the magnitude that 2–3
mo of bed rest has in altering the MHC phenotype. Moreover,
the alterations in MHC profile observed between the 60- and
90-day studies indicate that the additional 30 days of bed rest
between studies (90 vs. 60 days) had a greater impact on MHC
transformations.
Nutritional Countermeasure
The idea behind the nutrition countermeasure of increased
protein and leucine consumption was well supported by acute
muscle studies showing anabolic benefits for whole body and
muscle protein synthesis (8, 9, 12, 34). Further support came
from recent short-term (⬍30 days) bed rest studies showing
positive benefits for protein turnover and lean muscle mass
preservation (7, 37, 42, 43). However, the whole muscle (48)
and single muscle fiber data from the present 60-day bed rest
study suggest that the skeletal muscle benefits observed from
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Fig. 2. Summary of the vastus lateralis MHC IIa single-fiber parameters for
subjects in BR, BRN, or BRE groups before and after 60 days of bed rest. For
each variable (diameter, strength, speed, and power), the percent change is
shown.
acute metabolic studies and short-term bed rest studies do not
extend to longer duration bed rest. Interestingly, the nutrition
group lost significantly more thigh muscle mass than the
control group (48). At the single fiber level, the nutrition group
had a reduction in fiber diameter that was 5% greater than the
control group in both MHC I and IIa muscle fibers. Although
the difference in single fiber diameter between the nutrition and
control groups was not significant, it does agree with the whole
muscle mass data. Most likely, the additional 5% loss in fiber
diameter collectively contributed to the overall loss in thigh
muscle mass. On the basis of findings in the present study at
the whole muscle and single fiber level, nutrition alone appears
to exacerbate the loss in thigh muscle mass.
Our findings are in slight contrast to a recent study by
Paddon-Jones et al. (37), who reported a benefit for leg muscle
mass (using dual-energy X-ray absorptiometry) when an amino
acid and carbohydrate supplementation was consumed during
28 days of bed rest. In a subgroup of subjects from the same
study, vastus lateralis MHC I and IIa fiber diameters were not
protected by the nutritional supplementation (19). This discrep-
ancy between the whole muscle and single fiber data may be a
product of the techniques and specificity to assess changes in
muscle size, since the whole muscle measures did not isolate
the thigh muscles. These authors also reported that whole
muscle performance was significantly reduced in the nutrition
group, although not to the same degree as in the control group
(37). The offset in muscle performance between the nutrition
and control groups may have been related to the elevated
Table 7. Comparison of vastus lateralis single muscle fiber
size and contractile function changes between 60- and
90-day bed rest studies
MHC I Fibers MHC IIa Fibers
60 Day 90 Day 60 Day 90 Day
(Women) (Men) (Women) (Men)
Diameter, ␮m ⫺14% ⫺15% ⫺16% ⫺8%
Po, mN ⫺35% ⫺47% ⫺31% ⫺25%
Vo, FL/s ⫺12% ⫺21% ⫺9% ⫺6%
Power, ␮N 䡠 FL⫺1 䡠 s⫺1 ⫺42% ⫺55% ⫺28% ⫺25%
The 90-day bed rest data are from Trappe et al. (46).
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 VASTUS LATERALIS CONTRACTILE FUNCTION WITH BED REST AND CONCURRENT EXERCISE
shortening velocity of the MHC IIa fibers (reported significant
at the P ⬍ 0.1 level) relative to pre-bed rest (19). Although we
did not obtain muscle biopsies during the midpoint of the
60-day bed rest, which would provide a time point for direct
cellular comparison, we did find similar losses in the nutrition
and control groups’ thigh muscle mass (using MRI) on day 29
with a continued reduction in thigh muscle mass by day 59 of
bed rest period in the present study (48).
Gender Comparisons
One of the novel aspects of this investigation was the
inclusion of female volunteers. To our knowledge, potential
gender differences in skeletal muscle to unloading have been
relatively unexplored. Although of different study durations,
the present 60-day study provides some insight to changes in
skeletal muscle compared with our previous involvement in a
90-day bed rest study (46). A comparison of changes in MHC
I and IIa fiber size and contractile function from the control
subjects of the 60-day study (women) and the 90-day study
(men) are shown in Table 7. Perhaps the most interesting
aspect to note was the change in fiber size with bed rest in the
men and women. The men had a greater magnitude of atrophy
in the MHC I fibers (⫺15%) than in the MHC IIa fibers
(⫺8%), which was in agreement with previous animal studies
(18) showing a preferential atrophy of MHC I fibers during
unloading. In contrast, the women had a similar degree of
atrophy in both fibers types (⫺14 and ⫺16%). Furthermore,
the changes noted in muscle atrophy and contractile function in
the women occurred with 30 fewer days of bed rest.
The changes in vastus lateralis fiber diameter corroborate
the changes observed in whole muscle thigh mass. Using
MRI, we found that the women lost more thigh muscle mass
during 60 days of bed rest (48) than men during 90 days of
bed rest (3). Given that men and women had a similar
magnitude of atrophy in the MHC I fibers with bed rest, the
data point to the greater atrophy of the MHC IIa fibers as a
possible explanation for the additional thigh muscle mass
loss in women. This concept is supported by data showing
that old women have smaller MHC IIa fibers than old men
(31, 45). The reason for fiber-specific atrophy with unload-
ing and aging between men and women is unknown and
warrants further investigation.
Summary and Practical Applications
This study shows that the concurrent training program of
resistance (2–3 days/wk) and aerobic (3– 4 days/wk) exer-
cise was protective of the thigh muscle during 60 days of
bed rest in women. This finding is supported by the main-
tenance of whole muscle and single muscle fiber parameters
(MHC I and IIa) during the bed rest period. These data
indicate that, in light of the difficulty in protecting slow-
twitch muscle fibers by resistance training alone during
prolonged unloading (2, 3, 46), concurrent resistance and
aerobic training protocols should be considered as a viable
countermeasure for the upper leg of crewmembers during
long-duration space travel.
This 60-day bed rest study was the first dedicated to skeletal
muscle responses in females and provides a detailed profile of
muscle contractile function at the cellular level. The large
decline in fiber size and function in the nonexercisers was
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1249
massive by clinical standards and has serious ramifications for
metabolic health and mobility for individuals forced into long-
term bed rest situations. The nutrition countermeasure did not
provide an ergogentic benefit for single muscle fiber size and
function during bed rest, which is supported by the whole
muscle findings from these same women (48). This informa-
tion should be useful to the international space community and
the health care industry working with patients stricken with
extended bed rest.
ACKNOWLEDGMENTS
The authors thank Per Tesch, PhD, and Bjorn Alkner, MD, PhD, for
expertise and assistance with the resistance-training device and program. We
also thank the personal trainers (Marius Dettmer, Connie Fischer, Alex
Kowanz, Bjorn Redlich, and Nicolas Sinanan) and their supervisor (Casey
Pruett), medical oversight personnel (Dr. Arnaud Beck and Pascale Cabrol),
project managers (Dr. Peter Jost and Marie-Pierre Bareille), and muscle biopsy
operator (Dr. Jacques Mercier) for efforts with this investigation. We appre-
ciate Dr. Hargens’ team and Dr. Biolo’s team for their leadership with the
aerobic and nutritional countermeasures, respectively. We also give a special
thank you and appreciation to all the female volunteers from across Europe
who dedicated themselves to this space medicine project.
GRANTS
The study WISE-2005 was sponsored by the European Space Agency, the
US National Aeronautics and Space Administration (NASA), the Canadian
Space Agency, and the French “Centre National d’Etudes Spatiales,” which
has been the “Promoteur” of the study according to French law. The study was
performed by MEDES, Institute for Space Physiology and Medicine, in
Toulouse, France.
This phase of the investigation was supported by NASA Grant
NNJ04HF72G to S. Trappe and T. Trappe.
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