Bradley C. Nindl, Kevin R. Rarick, John W. Castellani, Alexander P.

Tuckow,
John F. Patton, Andrew J. Young and Scott J. Montain
J Appl Physiol 100:120-128, 2006. First published Sep 1, 2005; doi:10.1152/japplphysiol.01415.2004

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J Appl Physiol 100: 120 –128, 2006.
First published September 1, 2005; doi:10.1152/japplphysiol.01415.2004.
Altered secretion of growth hormone and luteinizing hormone after 84 h of
sustained physical exertion superimposed on caloric and sleep restriction
Bradley C. Nindl,1 Kevin R. Rarick,1 John W. Castellani,2 Alexander P. Tuckow,1
John F. Patton,1 Andrew J. Young,3 and Scott J. Montain3
1
Military Performance Division, 2Thermal and Mountain Medicine Division, and 3Military Nutrition
Division, United States Army Research Institute of Environmental Medicine, Natick, Massachusetts
Submitted 23 December 2004; accepted in final form 24 August 2005
Nindl, Bradley C., Kevin R. Rarick, John W. Castellani, Alex-
ander P. Tuckow, John F. Patton, Andrew J. Young, and Scott J.
Montain. Altered secretion of growth hormone and luteinizing hormone
after 84 h of sustained physical exertion superimposed on caloric and
sleep restriction. J Appl Physiol 100: 120 –128, 2006. First published
September 1, 2005; doi:10.1152/japplphysiol.01415.2004.—The pul-
satile release of growth hormone (GH) and luteinizing hormone (LH)
from the anterior pituitary gland is integral for signaling secretion of
insulin-like growth factor (IGF)-I and testosterone, respectively. This
study examined the hypothesis that 84 h of sustained physical exertion
with caloric and sleep restriction alters the secretion of GH and LH.
Ten male soldiers [22 yr (SD 3), 183 cm (SD 7), 87 kg (SD 8)] had
blood drawn overnight from 1800 to 0600 every 20 min for GH, LH,
and leptin and every 2 h for IGF-I (total and free), IGF binding
proteins-1 and -3, testosterone (total and free), glucose, and free fatty
acids during a control week and after 84 h of military operational
stress. Time-series cluster and deconvolution analyses assessed the
secretion parameters of GH and LH. Significant results (P ⱕ 0.05)
were as follows: body mass (⫺3%), fat-free mass (⫺2.3%), and fat
mass (⫺7.3%) declined after military operational stress. GH and LH
secretion burst amplitude (⬃50%) and overnight pulsatile secretion
(⬃50%), IGF binding protein-1 (⫹67%), and free fatty acids (⫹33%)
increased, whereas leptin (⫺47%), total (⫺27%) and free IGF-I
(⫺32%), total (⫺24%) and free testosterone (⫺30%), and IGF bind-
ing protein-3 (⫺6%) decreased. GH and LH pulse number were
unaffected. Because GH and LH positively regulate IGF-I and testos-
terone, these data imply that the physiological strain induced a certain
degree of peripheral resistance. During periods of energy deficiency,
amplitude modulation of GH and LH pulses may precede alterations
in pulse numbers.
hormone pulsatility; insulin-like growth factor-I; soldiers; leptin;
testosterone
THE ENDOCRINE RESPONSE TO sustained physical exertion super-
imposed on caloric and sleep restriction serves to provide
adequate fuels to meet metabolic needs for such physiological
processes as tissue repair, regeneration, and recovery (5, 7, 20).
In particular, the somatotropic [i.e., growth hormone (GH)/
insulin-like growth factor (IGF)-I] and pituitary-testicular [i.e.,
luteinizing hormone (LH)/testosterone] axes mediate many
metabolic and anabolic processes during altered energy states
(15, 16, 24, 32). The pulsatile release of GH and LH from the
anterior pituitary gland is integral for the trophic effects that
these hormones exert on IGF-I and testosterone, respectively
(35, 36). The manner in which anterior pituitary hormones are
released and delivered to target tissues is more biologically
Address for reprint requests and other correspondence: B. C. Nindl, Military
Nutrition Division, United States Army Research Institute of Environmental
Medicine, Natick, MA 01760 (e-mail: bradley.nindl@us.army.mil).
120
significant than the overall mean hormonal concentrations was
well illustrated by Isgaard et al. (17), who demonstrated that
GH administered in a pulsatile manner elicited greater in-
creases in IGF-I than GH administered in a continuous manner.
Because of the episodic release pattern of GH and LH, multiple
time-point measures are necessary to fully characterize the
trophic effects these hormones may have (18, 34, 35). Al-
though alterations in IGF-I and testosterone are known to be
Downloaded from jap.physiology.org on April 26, 2009
mediated by fluctuations in GH and LH, few studies have
concomitantly measured these hormones, especially in healthy
subjects exposed to multiple physiological stressors. Addition-
ally, recent work has suggested that leptin may also serve as an
integrator of neuroendocrine secretion of GH and LH (5, 24,
25). Information concerning the pulse parameters (i.e., fre-
quency, amplitude, etc.) of GH and LH is important to under-
stand when delineating central vs. peripheral mechanisms un-
derlying alterations in circulating endogenous hormones. Al-
though prior pulsatility programs (i.e., Pulsar, etc.) were
limited in that they provided only descriptive information
about hormone concentration peaks, recent advances in time
series software analysis now allow accurate determination of
underlying glandular hormonal secretion rate and half-life from
measurements obtained from the systemic circulation (34 –36).
Military operational scenarios represent a unique paradigm
in which to study endocrine responses to metabolic stress.
Soldiers, analogous to emergency rescue personnel (i.e., wild-
life firefighters, medical response teams, etc.) and ultra endur-
ance athletes (i.e, cyclists during the Tour de France, partici-
pants in the Eco challenge, etc.), are often required to perform
heavy sustained physical exertion in the face of nutritional and
sleep deprivation. Prior work on military populations has
shown that continued and prolonged exposure to such physio-
logical strain can lead to a suppression of circulating anabolic
growth factors, losses of lean body mass, increased suscepti-
bility to disease and infections, and diminished physical and
cognitive performance capabilities (4, 8, 9, 19, 27). For exam-
ple, Friedl et al. (9) extensively evaluated the effects of 62 days
of United States Army Ranger training during which young
healthy soldiers average losses of 15% in body mass, 7% in
fat-free mass, 65% in fat mass, and 20% decline in strength and
power, with 65 and 86% declines in IGF-I and testosterone,
respectively. The causal mechanisms underlying such over-
reaching and overtraining phenomena, particularly the short-
term endocrine and metabolic adaptations that may precede
critical functional impairments (i.e., chronic fatigue syn-
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.
http://www. jap.org
 ALTERED GH AND LH SECRETION
drome), are still poorly understood. Establishing a greater insight
into the fundamental origin of such processes could lead to the
identification of suitable “biomarkers” that once validated
could lead to early diagnostic and intervention strategies aimed
at buffering and sustaining peak physical performance.
Although much of the prior research on overtraining has
concentrated on expedient, “snapshot” biochemical measures
in the periphery, we hypothesized that alterations in the neu-
roendocrine release of GH and LH from the anterior pituitary
gland would also potentially represent a regulatory mechanism
in the early phase adaptational process after exposure to mul-
tiple physiological stressors. The purpose of the present study
was to further characterize the physiological consequences of
short-term military operational stress on the secretory patterns
of GH and LH release as analyzed by deconvolution algorithms
by obtaining serial blood samples (every 20 min) drawn for
12 h overnight from soldiers before and after 4 days of military
operational stress. The level of caloric restriction, sleep depri-
vation, and prolonged work were chosen to model current
“real-world” scenarios expected by the United States military
121
control week. The following week (military operational stress), the
second 96-h block was performed. During the military operational
stress week, the subjects performed nearly continuous physical work.
Sleep was restricted to two 1-h sleep periods per day. Food was
limited to one meal, ready-to-eat (1,200 –1,300 kcal/meal), and an
additional small meal per day for a total caloric intake of ⬃1,600
kcal/day.
Field training consisted of the performance of military relevant
tasks and military basic skill training. It included basic patrolling,
combat drills, road marches, land navigation, a litter obstacle course,
and a confidence course (refer to Table 1) (31). Physical activity and
movement were recorded every minute by a wrist-worn actigraph
(Mini Mitter, Bend, OR, and Precision Control Designs, Ft. Walton
Beach, FL). Sleep time was calculated by adding the time periods in
which no activity signal was measured. Water during the field training
was consumed ad libitum.
Subjects. Fourteen healthy male soldiers volunteered to participate
in the study. Three injuries and one illness prevented four of these test
subjects from completing the study. The data from the remaining 10
volunteers [22 yr (SD 3), 183 cm (SD 7), 87 kg (SD 8)] are presented
in this paper. The experiment was approved by the appropriate
Institutional Scientific Review and Human Use Review Committees.

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in high-intensity combat situations. In addition, we concomi-
tantly measured the downstream anabolic mediators of these
signaling peptides: testosterone and IGF-I, IGF binding pro-
teins (BPs)-1 and -3, and other metabolic markers (i.e., leptin,
free fatty acids, and glucose).
METHODS
Experimental design. This study utilized a within-subjects repeated-
measures design consisting of two 96-h testing blocks (see Table 1).
The results of the physical performance tests performed on the
morning of day 1, day 3, and day 4 of each test block, as well as cold
stress test on day 4, have been reported previously (4, 30). The initial
block (control week) included the experimental tests but no other
scheduled activities. Sleep and food intake were ad libitum during the
The subjects were briefed on the study procedures, and written
consent was obtained before study participation.
Body composition. Body composition was assessed by whole body
dual-energy X-ray absorptiometry. Total body estimates of percent
fat, bone mineral density, and bodily content of bone, fat, and
nonbone lean tissue were determined using manufacturer-described
procedures and supplied algorithms (Total Body Analysis, version
3.6, Lunar, Madison, WI). Precision of this measurement is better than
⫾0.5% body fat. Scanning was in 1-cm slices from head to toe using
the 20-min scanning speed (8).
Overnight serial blood sampling. On two separate occasions (see
Table 1), subjects had blood drawn overnight in a metabolic research
laboratory. The subjects reported to the laboratory at 1630, were
served a dinner meal at 1700, and then had an indwelling catheter
inserted at 1745. Beginning at 1800, blood was serially sampled every

Table 1. Timeline and activities for the experimental protocol

Time Day 1 Day 2 Day 3 Day 4 Day 5

0100 Road march Road march Road march 12-h hormonal profile
0200 analysis*
0300 Travel/sleep
0400 Sleep Sleep
0500 Break Unload truck/break Break
0600 Blood draw* Confidence course Blood draw* Blood draw* Body composition
0700 Cognitive and Cognitive and Cognitive and
0800 marksmanship testing* marksmanship testing* marksmanship testing*
0900 Physical performance Break Physical performance Physical performance
1000 testing* Battle drills testing* testing* Debrief
1100 Travel to field site Break Cold test*
1200 Set up command post Hot walk*
1300
1400 Break
1500 Land navigation course Travel Physical training
1600 Litter carry
1700 Break
1800 Break 12-h hormonal profile
1900 Break Battle drills Light training analysis*
2000 Battle drills Pack command post
2100 Sleep
2200 Sleep Road march Sleep
2300 Road march Road march
2400
*Testing blocks that were performed during both the control and military operational stress weeks; all other areas were activities that were only performed
during the military operational stress week.

J Appl Physiol • VOL 100 • JANUARY 2006 • www.jap.org

122 ALTERED GH AND LH SECRETION
20 min for GH, LH, and leptin (36 samples total) and every 2 h for the
IGF-I system components, testosterone, glucose, and free fatty acids
(6 samples total) until 0600 the next morning. Blood sampling
followed the same procedure following the military operational stress
paradigm. To facilitate familiarization with the facility and minimize
sleep disruptions, all subjects slept in the metabolic laboratory the
night before the control overnight serial blood draw. Conditions were
mimicked exactly to include the taping of a catheter near the antecu-
bital vein for the night. During each overnight trial, subjects were
allowed to move freely within the laboratory. Lights were turned off
at 2200, and the TV was turned off at 2300. At bedside, a registered
nurse and research technicians unobtrusively performed the serial
blood draws throughout the night. Blood was collected in glass
vacutainers, allowed to clot on ice, and centrifuged for 30 min at 1,500
g at 4°C. After centrifugation, serum was aliqoted into the appropriate
Eppendorf tubes, flash frozen in liquid nitrogen, and stored at ⫺80°C
until later analysis.
Biochemical assays. GH, LH, and leptin concentrations were mea-
sured using a two-site immunoradiometric assays (Diagnostic Sys-
tems Laboratories, Webster, TX). The sensitivity and intra-assay
variances for these assays were 0.01 ng/ml and 3.9%, 0.12 mIU/ml
and 6.8%, and 0.10 ng/ml and 3.7% for GH, LH, and leptin, respec-
tively. Total IGF-I, free IGF-I, IGFBP-1, and IGFBP-3 concentrations
were determined by a two-site immunoradiometric assays (Diagnostic
System Laboratories). The sensitivity of these assays was 2.06, 0.03,
0.33, and 0.05 ng/ml for total IGF-I, free IGF-I, IGFBP-1, and
IGFBP-3, respectively. Total and free testosterone concentrations
were determined by a radioimmunoassay (Diagnostic Systems Labo-
ratories). The sensitivity of these assays was 0.08 ng/ml and 0.18
pg/ml, respectively. The intra-assay variances were both ⬍4.0%.
Radioimmunoassays were performed on a Cobra gamma counter
(Packard Instruments, Downers Grove, IL). Serum glucose concen-
trations (Bio-Chem Laboratory Systems, Lakewood, NJ) and free
fatty acids (Wako Chemicals, Richmond, VA) were measured accord-
ing to manufacturer’s instructions on an ATAC 8000 (Elan Diagnos-
tics, Smithfield, RI). The intra-assay variances were all ⬍7%. Sam-
ples were run in duplicate for all assays. All samples for a given
subject were run in the same assay batch to eliminate interassay
variance.
Cluster analysis. The cluster analysis program was used to analyze
the overall mean GH, LH, and leptin concentrations, 12-h integrated
area under the curve via trapezoidal rule, and characteristics of
concentration “peaks” (18, 35, 36). The cluster algorithm searched for
significant increases and decreases among data points in a series via
pooled t-tests. Two points were used for the determination of a peak
and one point to establish a nadir. A peak was defined as a series of
concentrations that demonstrated an increase over time followed by a
decrease, with the additional requirement that a nadir (i.e., a decrease
followed by an increase) was present on each side of the peak. Cluster
analysis was utilized to provide descriptive information regarding
observed concentrations as a function of time, such as the number of
peaks in 12 h, the mean interval between peaks, the mean peak width
Table 2. Hormone concentration parameters from Cluster 7 program for LH, GH, and leptin
during the control and military stress conditions
LH, IU/ml
Parameter Control Military stress P Control
Mean, min 3.7 (1.0) 5.4 (1.8) 0.014* 0.6 (0.5)
Total area, min 2,534.1 (795.7) 3,633.1 (1276.5) 0.014* 426.7 (365.4)
Peak height, min 5.5 (1.8) 8.6 (2.5) 0.029* 1.8 (1.7)
Peak number 3.5 (1.2) 3.2 (1.6) 0.530 3.5 (1.1)
Peak width, min 128.3 (27.0) 136.8 (45.1) 0.642 151.4 (60.4)
Peak area, min 165.1 (79.1) 284.0 (154.5) 0.176 148.5 (187.8)
Values are means (SD). GH, growth hormone; LH, luteinizing hormone. *Significant difference between conditions (P ⬍ 0.05).
J Appl Physiol • VOL 100 • JANUARY 2006 •
(i.e., duration), the mean peak height (i.e., amplitude), and the area
under peaks.
Deconvolution analysis. Multiple-parameter analysis was used to
estimate the secretion and elimination characteristics of the hormones
based on the assay-measured concentrations. This method used a
convolution integral that is solved by nonlinear least squares param-
eter estimation (18, 35, 36). For this analysis, we used the same data
file that was configured and used for the Cluster program. The
hormone concentration data was initially deconvolved using the
Pulse2 program to estimate the number and position of secretion
bursts. Variable basal secretion and an estimated half-life, chosen to
produce a convolved curve that closely approximated the actual
concentration data, were selected as the input parameters for Pulse2.
Deconvolution analysis was further carried out with multiple-param-
eter fitting at 95% joint statistical confidence intervals for all calcu-
lated secretory burst variables. Half-life, secretion burst number,
interval between bursts, burst area, burst amplitude, amplitude of
largest burst, basal secretion per minute, overnight basal secretion
(basal secretion ⫻ 720 min), overnight pulsatile secretion (burst
area ⫻ secretion burst number), and total overnight secretion (over-
night basal secretion ⫹ overnight pulsatile secretion) were determined
for GH and LH.
Downloaded from jap.physiology.org on April 26, 2009
Statistical analyses. All data are presented as means (SD). A
two-way repeated ANOVA with repeated measures was employed for
statistical analyses. Where appropriate, a Tukey’s post hoc follow-up
test was used. For the secretion characteristics, a dependent t-test was
used to compare the two means. All statistical analyses were per-
formed with Statistica software packages (StatSoft, Tulsa, OK). An
alpha level of 0.05 was used for all statistical evaluations.
RESULTS
Body composition. Subjects consumed an average of 1,653
kcal/day (SD 25) [225 g carbohydrate (SD 6), 54 g fat (SD 2),
69 g protein (SD 3)] and slept a total of 6.2 h (SD 0.4) during
the entire 84-h experimental study period as assessed by
actigraphy. Energy expenditure, estimated from distance tra-
versed and predicted energy cost of the activities during the
course, was ⬃4,000 – 4,500 kcal/day. Body mass [control: 84.8
(SD 3.7); military operational stress: 82.2 (SD 3.5)], fat free
mass [control: 69.0 (SD 2.7); military operational stress: 67.7
(SD 2.5)], fat mass [control: 15.8 (SD 1.7); military operational
stress: 14.5 (SD 1.8)], and percent body fat [control: 18.4 (SD
1.3); military operational stress: 17.4 (SD 1.5)] all significantly
declined (P ⬍ 0.05) during the experimental condition.
Cluster analysis of LH, GH, and leptin. Table 2 presents the
LH, GH, and leptin concentration parameters estimated by
cluster analysis. LH, GH, and leptin mean overnight concen-
tration values and total area under the curve were significantly
different (P ⬍ 0.05) between the control and military opera-
GH, ng/ml Leptin, ng/ml
Military stress P Control Military stress P
0.9 (0.4) 0.023* 3.8 (2.6) 2.0 (1.4) 0.009*
653.0 (284.2) 0.027* 2,555.9 (1,771.2) 1,333.8 (958.2) 0.009*
2.9 (1.7) 0.083 4.4 (2.9) 2.3 (1.5) 0.005*
3.0 (0.9) 0.170 3.1 (1.6) 3.3 (2.2) 0.931
146.5 (46.0) 0.853 185.6 (121.3) 106.0 (75.3) 0.107
182.9 (114.3) 0.584 300.4 (554.9) 56.7 (53.9) 0.262
www.jap.org
 ALTERED GH AND LH SECRETION 123
tional stress conditions, respectively. LH peak height was
significantly higher after military operational stress, whereas
leptin peak height was lower. There were no statistical differ-
ences found for LH, GH, or leptin between the control and
military stress conditions for number of peaks, width of peaks,
or area under a peak. GH and LH cluster analysis graphs for
two representative subjects are presented in Figs. 1 and 2.
Figure 3 displays the composite hormonal concentrations for
all subjects in both the control and military stress conditions for
GH, LH, and leptin.
Deconvolution analysis of LH and GH. The LH and GH
secretory dynamics as estimated by deconvolution analysis are
given in Table 3. LH half-life, number of bursts, amplitude of
largest burst, basal secretion rate, overnight basal secretion, or
total overnight secretion (basal ⫹ pulsatile) was similar (P ⱖ
0.05) between conditions. However, LH overnight pulsatile
secretion, area under bursts, amplitude of bursts, and interval
between bursts were greater after the military operational stress
condition. Thus the increase in mean LH concentration was
accounted for by an increase in the amount of hormone

Downloaded from jap.physiology.org on April 26, 2009

secreted per burst and not an increase in the number of bursts
or any change in the basal secretion rate.

Fig. 2. Cluster and deconvolution analyses of luteinizing hormone (LH)

(subjects C and D; A and B, respectively) for the 12-h overnight sampling
period. Top: hormone concentrations from cluster analysis for both control
(left) and military stress (right) conditions. Bottom: corresponding secretory
bursts from deconvolution analysis.

Mean GH concentration was significantly increased after

Fig. 1. Cluster and deconvolution analyses of growth hormone (GH) (subjects
A and B; A and B, respectively) for the 12-h overnight sampling period. Top:
hormone concentrations from cluster analysis for both control (left) and
military stress (right) conditions. Bottom: corresponding secretory bursts from
deconvolution analysis.
J Appl Physiol • VOL
military stress compared with the control condition. The in-
crease in mean GH concentration was accompanied by an
increase in the following secretion parameters: area under
bursts, amplitude of bursts, basal secretion rate, overnight basal
secretion, overnight pulsatile secretion, and total overnight
secretion. GH half-life, number of bursts, interval between
bursts, and amplitude of largest burst were similar between the
control and military operational stress conditions. Representa-
tive GH and LH profiles of deconvolution analysis for two
subjects are presented in Figs. 1 and 2.
IGF-I system, testosterone, glucose and free fatty acids.
Figures 4 – 6 display the overnight metabolic and hormonal
responses sampled every 2 h overnight for the control and
the military stress conditions. Table 4 presents a summary
for the overall main effects means for these variables. Total
IGF-I, free IGF-I, IGFBP-3, total testosterone, and free
testosterone were significantly lower after military stress,
whereas IGFBP-1 and free fatty acids were significantly ele-
vated after military stress. Glucose concentrations were similar
for both conditions.
100 • JANUARY 2006 • www.jap.org
124 ALTERED GH AND LH SECRETION

data indicate that the experimental paradigm was metabolically

challenging as evidenced by the estimated daily caloric deficit
(⬃2,500 –2,900 kcal), the loss in body mass (⬃3.0%), and the
limited amount of sleep attained (total of 6.2 h). Marked
alterations in the secretory release patterns (i.e., amplifications)
of GH and LH and the concentrations of other metabolic and
hormonal modulators (i.e., IGF-I, testosterone, leptin, free fatty
acids) as well as body composition parameters were observed.
The fact that short-term (4 days) military operational stress can
have striking effects on the neuroendocrine system raises
questions for future studies concerning the potential adverse
impact of long-term military operational stress (i.e., overseas
deployments lasting 6 –12 mo) on soldier physiology and the
subsequent health and fitness of our military personnel.
This study used a rigorous serial blood sampling scheme and
subsequent time-series deconvolution analyses to assess GH
and LH secretion patterns. These data expand on prior research
that relied on one time-point blood samples to glean informa-
tion on the endocrine responses to stress that are subject to
episodic and circadian release patterns (9, 13, 27). The phys-

Downloaded from jap.physiology.org on April 26, 2009

Fig. 3. Mean concentration of all subjects over the 12-h sampling period.
Samples were taken every 20 min for a total of 36 blood draws as represented
on the x-axis. Main effect mean concentrations of GH (A), LH (B), and leptin
(C) after the military stress condition are statistically different (P ⱕ 0.05) from
control for all hormones (also see Table 2).
DISCUSSION
In an effort to yield novel insights into the early phase
neuroendocrine responses to physiological strain, we modeled
military operational field training (i.e., 4 days of sustained
physical activity, caloric restriction, and sleep deprivation) to
examine overnight somatotrophic hormonal responses. Our
iological significance of studying the pulsatile release of these
hormones lies in the fact that the episodic release pattern is
thought to be integral in eliciting optimal agonist function at
the cellular level (17, 34 –36). The salient finding emerging
from this study was an amplification of GH and LH secretion
with a concomitant decline in IGF-I and testosterone. Because
GH and LH are known to positively regulate IGF-I and
testosterone, these data imply that the physiological strain
experienced in this study induced a certain degree of peripheral
resistance.
GH/IGF-I axis. The mean GH concentration and total area
were 50% greater after military operational stress as assessed
by cluster analysis. More detailed analysis of the secretory
dynamics of GH indicated an amplification in the following
pulsatility parameters: area (43%) and amplitude (50%) of
bursts, basal secretion rate (63%), overnight basal (54%) and
pulsatile (55%) secretion rate, as well as total overnight secre-
tion (55%). GH half-life and the number of bursts were
unaffected. Although the magnitude of these responses is
substantial, they are considerably less than observed after
similar periods of total fasting. For example, Ho et al. (16)
reported a 100% increase in GH peak amplitude after 5 days of
fasting in healthy young men, and Hartman et al. (15) reported
a 400 –500% increase in GH production rate and a 200%

Table 3. Hormone secretion parameters from deconvolution analysis for LH and GH

during the control and military stress conditions
LH, IU/ml GH, ng/ml

Parameters Control Military Stress P Control Military Stress P

Half-life, min 45.15 (8.66) 51.77 (9.96) 0.095 16.36 (4.33) 18.52 (4.58) 0.233
Number of bursts/12 h 6.00 (0.89) 5.67 (1.03) 0.363 4.62 (1.51) 5.62 (1.30) 0.050
Interval between bursts, min 109.30 (15.54) 120.31 (15.27) 0.049* 96.06 (37.99) 95.48 (28.92) 0.978
Area under bursts, min 3.86 (2.00) 5.90 (2.65) 0.028* 3.04 (1.47) 4.31 (2.16) 0.044*
Amplitude of bursts, min 0.13 (0.07) 0.21 (0.08) 0.016* 0.10 (0.05) 0.16 (0.09) 0.040*
Amplitude of largest burst, min 0.23 (0.11) 0.32 (0.10) 0.100 0.24 (0.15 0.36 (0.22) 0.186
Basal secretion rate, min 0.03 (0.01) 0.03 (0.01) 0.797 0.001 (0.000 0.002 (0.001) 0.020*
Overnight basal secretion, 12 h 19.22 (6.64) 18.46 (7.71) 0.797 0.80 (0.19 1.29 (0.55) 0.020*
Overnight pulsatile secretion, 12 h 22.78 (11.85) 33.20 (15.78) 0.021* 15.14 (11.09 23.28 (10.16) 0.029*
Total overnight secretion, 12 h 42.00 (17.62) 51.66 (20.94) 0.107 15.94 (11.13) 24.57 (10.59) 0.027*
Values are means (SD). *Significant difference between conditions (P ⬍ 0.05).

J Appl Physiol • VOL 100 • JANUARY 2006 • www.jap.org

 ALTERED GH AND LH SECRETION
increase in GH mean burst mass in older subjects after a 48-h
fast. More recently, Maccario et al. (24) sampled blood every
30 min from 0800 to 1600 after a 36-h fast and observed a
mean increase in GH from 0.5 ng/ml in the fed condition to 6.2
ng/ml in the fasted condition, whereas Chan et al. (5) sampled
blood every 15 min for 24 h after a 72-h fast in young, lean
men and reported ⬃114% increases in mean GH concentration
and integrated area. The obvious explanation for dissimilarity
between our results and those reported above was that, al-
though our subjects were exposed to a significant energy
deficit, they were provided daily meals (⬃1,600 kcal/day).
Friedl et al. (9) have previously reported that even small
increases in caloric consumption can have significant effects on
restoring hormonal concentrations toward baseline values in
soldiers exposed to field training. The results from the present
study also differed from the literature in that there were no
significant differences in the number of pulses observed,
whereas pure fasting studies have reported increases up to
130% in pulse frequency (5, 15, 24). These findings could
perhaps be interpreted to suggest that, during periods of energy
deficiency, amplitude modulation of GH pulses precedes alter-
ations in pulse number.
IGF-I and its family of BPs are well recognized as metabolic/
growth/maintenance modulators during altered energy states
(1, 2, 26, 28, 29, 32). Our laboratory previously reported
responses of IGF-I molecular complexes during this same
J Appl Physiol • VOL
125
Fig. 4. Insulin-like growth factor (IGF)-I
system responses during the control and mil-
itary stress week measured bi-hourly from
1800 to 0600. A: total IGF-I. B: free IGF-I. C:
IGF binding protein (BP)-1. D: IGFBP-3.
Downloaded from jap.physiology.org on April 26, 2009
military operational field exercise as measured by morning
blood draws taken throughout the study (26). The present
results utilize a series of blood draws to expand on those
observations by showing the sustained impact on the IGF-I
system during the 12-h overnight period. Total, free IGF-I, and
IGFBP-3 were reduced after the military operational stress by
27, 32, and 6%, respectively. In contrast, IGFBP-1 increased
by 67%. IGF-I has been shown to decline by 67 and 70% after
5 and 10 days, respectively, of complete fasting in healthy
adults (6) and by ⬃50% after 14 days of United States Army
Ranger training (9). That nearly one-half the magnitude of
these reductions was achieved in the present study underscores
the severity of the caloric imbalance and metabolic strain
imposed in the present study and how rapidly IGF-I can
change. IGF-I secretion from the liver is the primary biological
targeted outcome for GH. The fact that IGF-I decreased even in
the presence of amplified GH release suggests that the liver
was “GH resistant” (32). The underlying mechanism may be
associated with an impairment of the JAK-STAT signaling
pathway. Beauloye et al. (3) reported a decrease in GH-
stimulated tyrosine phosphoryaltion of JAK2, GHR, and
STAT5, without significant changes in their protein content in
rat liver after a 48-h fast. Elevated cortisol (not measured in the
present study) could also have contributed to a decreased
peripheral GH sensitivity. As pointed out by Chan et al. (5), the
adaptive value of increased GH and decreased IGF-I would be
100 • JANUARY 2006 • www.jap.org
126 ALTERED GH AND LH SECRETION

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Fig. 6. Glucose (A) and free fatty acids (B) during the control and military
Fig. 5. Testosterone responses during the control and military stress week stress week measured bi-hourly from 1800 to 0600.
measured bi-hourly from 1800 to 0600. A: total testosterone response. B: free
testosterone response. Opstad (31), who reported a 33% decrease in LH after a 5-day
military operational period as measured from a one-time morn-
ing blood sample. Consistent with the increase in overall mean
a diminishment of energy expenditure required for growth-
LH concentration, deconvolution analysis of LH showed in-
related processes while enabling GH to promote the mobiliza-
tion of alternative fuels through lipolysis.
Of the IGF-I system components measured, IGFBP-1 dem- Table 4. Means for main condition effects during the
onstrated the largest magnitude change. Frystyk et al. (10) control and military stress conditions
speculated that the IGFBP-1 increase may be viewed as a
Variable Control Military Stress P
protective mechanism by neutralizing the insulin-like hypogly-
cemic activity of IGF-I and serving to mobilize energy from Total IGF-I, ng/ml 156.8 (65.0) 114.1 (49.4) 0.004*
fats and protein. Furthermore, it has been suggested that low Free IGF-I, ng/ml 2.2 (1.3) 1.5 (0.8) 0.025*
levels of liver glycogen are linked to an increased secretion of IGFBP-I, ng/ml 22.1 (22.9) 37.0 (33.5) 0.007*
IGFBP-3, ng/ml 35.5 (8.7) 33.6 (9.9) 0.064
IGFBP-1 (21). IGFBP-1 remains an important regulatory BP to Total testosterone, ng/ml 5.5 (1.9) 4.2 (1.6) 0.002*
study relative to physiological stress as increased levels may be Free testosterone, pg/ml 17.9 (7.4) 12.5 (6.1) 0.001*
one mechanism whereby IGF-I bioactivity is adjusted to meet Free fatty acid, mEq/l 0.4 (0.2) 0.6 (0.3) 0.013*
metabolic needs. Glucose, mg/dl 90.4 (10.6) 93.2 (13.2) 0.406
LH/testosterone axis. The mean LH concentration increased Values are means (SD). *Significant difference between conditions (P ⬍
46% for the military stress condition. This is in contrast to 0.05).

J Appl Physiol • VOL 100 • JANUARY 2006 • www.jap.org

 ALTERED GH AND LH SECRETION
creases in burst area (53%), burst amplitude (64%), and over-
night pulsatile secretion (46%). Loucks et al. (22) have recently
reported that in women LH pulsatility is disrupted when energy
intake is below a threshold of 30 kcal䡠kg LBM⫺1 䡠day⫺1.
Similar to our findings, Loucks and Thuma (22) reported an
increase in burst amplitude (21%), but in contrast to our results,
they reported a slowing of the LH burst frequency (⫺16%) and
a maintenance of LH mean concentration. In our study, sub-
jects consumed an average of 23.5 kcal 䡠kg LBM⫺1 䡠day⫺1,
placing them well into a negative energy balance with a deficit
of ⬃2,500 –2,900 kcal/day and below the hypothesized thresh-
old of 30 kcal䡠kg LBM⫺1 䡠day⫺1 necessary for the disruption
of LH pulsatility in women (22). Deconvolution analysis re-
vealed the military stress condition to have a significantly
increased interval between bursts but a nonsignificant decline
(⫺8.6%) in LH burst frequency. The increased burst amplitude
and the maintained burst frequency resulted in an increased LH
mean concentration.
Despite enhanced release of LH, testosterone concentrations
decreased. During the 12-h overnight sampling period, total
and free testosterone concentrations were 24 and 30% lower
after the military stress condition, respectively. Decreased
levels of testosterone have also been observed in other studies
that investigated the effects of short-term military stress (9, 27,
31). Opstad (31) found a 47% decrease in morning testosterone
concentration after a 5-day military training course. Under
similar conditions, Gomez-Merino et. al. (11) found a 35%
decrease in morning testosterone concentrations. Both of these
studies employed a single time-point analysis. By considering
the circadian rhythm of testosterone, our study has extended
these findings by demonstrating that testosterone concentra-
tions are decreased and remain depressed overnight for a 12-h
period following the conclusion of a multiple-day military
stress condition.
Conceptually, a mechanism to maintain normal testosterone
concentrations could be an enhanced pulsatile release of LH.
Decreased testosterone concentrations relieve the inhibition of
the negative feedback loop at the hypothalamus/pituitary
gland, resulting in an enhanced release of gonadotropin-releas-
ing hormone and subsequently enhancing LH secretory dynam-
ics. For example, compared with younger men, older men tend
to possess greater LH secretion, which is hypothesized to
represent a biological attempt to maintain normal testosterone
concentrations (14). Endurance-trained athletes have also
shown decreased testosterone and increased LH concentrations
(11, 12). The literature with regard to acute exercise stress has
been equivocal. Tanaka et al. (33) have reported decreased
concentrations of testosterone and increased LH concentrations
for up to 2 days after a marathon and suggested that elevated
catecholamines may be partially responsible for the decreased
testicular sensitivity. We have previously reported decreased
LH secretion and decreased testosterone concentrations after
heavy resistance exercise in men. The fact that all subjects
were provided a small meal ⬃1 h before the overnight blood
draws may be of consequence in terms of interpreting the LH
data in that a single ad libitum meal has been reported to
stimulate LH pulsatility within hours in a number of different
species (25). Thus, although LH pulsatility is slowly restored in
women (23), there is evidence to suggest that gonadotropin-
releasing hormone neurons may rapidly respond to caloric
intake.
J Appl Physiol • VOL
127
Leptin/glucose/free fatty acid responses. Leptin concentra-
tions declined from 3.8 to 2.0 ng/ml (⬃47% decline) after
military stress. These declines were coincident with marked
reductions in fat mass (⬃8%) and increased concentrations of
circulating free fatty acids (⬃33%). This magnitude decrease
for leptin is consistent with the 44% decrease reported by
Maccario et al. (24) after 36 h of fasting, but less than the
⬃66% decline observed by Gomez-Merino et al. (11) after a
5-day military combat course. These changes (leptin and free
fatty acids) are reflective of substantial lipolytic activity (also
supported by the amplification of GH release). Also of interest
is the fact that the overall main effect mean for glucose concen-
trations was similar between the control (90.4 mg 䡠dl⫺1 䡠l⫺1) and
military stress (93.2 mg 䡠dl⫺1 䡠l⫺1) condition, suggesting that
glucose homeostasis was well defended in this paradigm.
Although not measured in the present study, other studies have
reported robust changes in cortisol after military stress (9), and
it is also probable that both cortisol and catecholamine eleva-
tions played a contribution in maintaining glucose concentra-
tions. The fact that the absolute losses in fat-free mass (1.3 kg)
Downloaded from jap.physiology.org on April 26, 2009
and fat mass (1.3) were similar suggests that amino acid fuel
partially contributed to gluconeogenesis.
Converging lines of evidence in sheep, rat, monkey, and
human models suggest that leptin acts as a peripheral signal to
mediate metabolic regulation of GH and LH secretion from the
anterior pituitary gland (5, 25). Leptin appears to prevent the
fasting-induced suppression of pulsatile release of LH secre-
tion. In a recent study that evaluated the effects of differing
doses of exogenously administered r-metHuLeptin in men
during 73 h of fasting and employing 24-h blood sampling,
Chan et al. (5) demonstrated a role for leptin in normalizing
starvation-induced neuroendocrine changes, notably the hypo-
thalamic-pituitary-gonadal axis. Chan et al. (5) hypothesized
that a threshold leptin level between 0.5 and 2.0 ng/ml may be
necessary for LH secretion. It is interesting to note that, in our
study, the leptin concentrations after military stress were 2.0
ng/ml and that there was not a decline in LH pulse frequency.
Although glucose concentrations have also been speculated to
act as a peripheral signal relaying information concerning
nutritional status and within the context of the dramatic hor-
monal changes observed (20), it is remarkable that glucose
homeostasis was well maintained. The overall physiological
and metabolic stress encountered in the present study appears
to fall below a threshold necessary to disrupt the maintenance
of normal glucose homeostasis.
In conclusion, our results demonstrate that brief periods (i.e.,
4 days) of intensive physical activity superimposed on energy
and sleep restriction induced amplifications in GH and LH
secretion (no change in pulse number, however), yet at the
same time resulted in decreases in hormones that these signal-
ing peptides are responsible for stimulating, namely IGF-I and
testosterone. That short-term military operational stress can
exert marked effects in neuroendocrine secretion patterns
raises important questions concerning the potential adverse
effects on longer-term military operational stress (i.e., 6- to
12-mo overseas deployments) on underlying physiology and
metabolism and the health status of military personnel.
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