Cell Stress & Chaperones (2006) 11 (2), 129–134
? Cell Stress Society International 2006
Article no. csac. 2006.CSC-158R
Human resting extracellular heat
shock protein 72 concentration
decreases during the initial
adaptation to exercise in a hot,
Helen C. Marshall, Richard A. Ferguson, and Myra A. Nimmo
Department of Applied Physiology, Faculty of Science, University of Strathclyde, Glasgow, G4 0QU, UK
Abstract Heat shock protein (Hsp) 72 is a cytosolic protein that also is present in the circulation. Extracellular Hsp72
(eHsp72) is inducible by exercise and is suggested to act as a danger signal to the immune system. The adaptive
response of eHsp72 to repeated exercise-heat exposures in humans remains to be determined. An intracellular animal
study found a reduced Hsp72 response, with no change in resting levels, during heat stress after a single day of
passive heat acclimation. The current study therefore tested whether adaptations in human eHsp72 levels would
similarly occur 24 hours after a single exercise-heat exposure. Seven males completed cycle exercise (42.5%˙VO2peak
for 2 hours) in a hot, humid environment (38?C, 60% relative humidity) on each of 2 consecutive days. Blood samples
were obtained from an antecubital vein before exercise and 0 hours and 22 hours postexercise for the analysis of
eHsp72. Exercise-heat stress resulted in enhanced eHsp72, with a similar absolute increase found on both days (day
1: 1.26 ng/mL [0.80 ng/mL]; day 2: 1.29 ng/mL [1.60 ng/mL]). Resting eHsp72 decreased from rest on day 1 to day
2’s 22-hour postexercise sample (P ? 0.05). It is suggested that the reduction in resting eHsp72 after 2 consecutive
exercise-heat exposures is possibly due to an enhanced removal from the circulation, for either immunoregulatory
functions, or for improved cellular stress tolerance in this initial, most stressful period of acclimation.
Heat shock proteins (Hsps) are produced in response to
a wide variety of stressors, including oxidative stress
(Salo et al 1991; Smolka et al 2000), glucose deprivation
(Febbraio et al 2002b), hyperthermia (Skidmore et al 1995;
Kregel and Moseley 1996; Fehrenbach et al 2001), and ex-
ercise (Skidmore et al 1995; Liu et al 1999; Noble et al
1999; Fehrenbach et al 2000a, 2000b). The most thermo-
sensitive and highly inducible Hsp belongs to the 70 kDa
family (Mizzen and Welch 1988) and is commonly known
as Hsp72. Primarily thought of as an intracellular protein
with cytoprotective functions, it also has been suggested
that Hsp72 has a functional role when released by a va-
riety of cells (Febbraio et al 2002a; Broquet et al 2003;
Correspondence to: Myra A. Nimmo, Tel: ?44 (141) 548 5791; Fax: ?44
(141) 548 5792; E-mail: email@example.com.
Received 19 August 2005; Revised 20 November 2005; Accepted 28
Hunter-Lavin et al 2004; Lancaster et al 2004) into the
circulation (eHsp72) (Pockley et al 1998). eHsp72 has
been suggested to have an immunological function
(Moseley 1998; Asea et al 2000; Bethke et al 2002; Campisi
and Fleshner 2003; Fleshner et al 2003) with the in vitro
attachment of eHsp72 to the surface of monocytes being
found to stimulate the production of several cytokines
(Asea et al 2000). Any change to the eHsp72 concentra-
tion may therefore have a subsequent effect on the cyto-
kine cascade and resulting inflammatory response. In ad-
dition, elevated levels of eHsp72 have been found in pa-
tients with pulmonary and renal vascular disease (Wright
et al 2000), and have been linked with a reduced risk of
coronary artery disease (Zhu et al 2003). It appears, there-
fore, that eHsp72 is an important molecular marker about
which little is known in conjunction with exercise-heat
stress in humans.
Prolonged exercise in a temperate environment has
Cell Stress & Chaperones (2006) 11 (2), 129–134
Marshall et al
been shown to be an adequate stressor to enhance the
concentration of eHsp72 (Walsh et al 2001; Lancaster et
al 2004; Fehrenbach et al 2005), the magnitude of which
is both exercise-intensity and duration dependent (Feh-
renbach et al 2005). After acute exercise, the concentration
of eHsp72 has been shown to return to resting levels
within 24 hours (Walsh et al 2001; Fehrenbach et al 2005).
An enhanced level of stress, as measured by intracellular
Hsp72 levels in human monocytes, has been found after
prolonged exercise in a high ambient temperature (Feh-
renbach et al 2001); however, the effect of exercise-heat
stress on eHsp72 has yet to be shown.
Repeated exposures to exercise-heat stress result in
many adaptations, the combination of which ultimately
improves thermal tolerance and athletic performance.
During acute heat stress, before the physiological adap-
tations of heat acclimation, the strain imposed on the hu-
man body is reduced through enhanced Hsp72 synthesis
(Moseley 1997). An in vitro study on mammalian cell
lines found an initial heat shock (43?C, 1.5 hours) to result
in an overall reduction in the amount of Hsp72 synthesis
during a subsequent heat shock (45?C, 30 minutes) (Miz-
zen and Welch 1988). This is supported by an in vivo
study in rats that showed a single day of passive heat
acclimation to result in a blunted rat heart Hsp72 increase
after heat stress (41?C, 2 hours) compared with no prior
acclimation (Maloyan et al 1999). However, the resting
Hsp72 level was found to remain unchanged in the initial
stage (1 or 2 days) of passive heat acclimation, in contrast
to the enhanced resting concentration found after long-
term heat acclimation (30 days) (Maloyan et al 1999). To
our knowledge, however, no literature is available on the
adaptation of resting eHsp72 or the eHsp72 response to
the early stages of heat acclimation in humans.
It is therefore hypothesized that eHsp72 will increase
after an acute exercise-heat stress with a blunted response
during a second consecutive, identical exposure. How-
ever, no change in resting eHsp72 is expected.
MATERIALS AND METHODS
Seven male subjects (mean [SD]; age, height, body mass,
and peak oxygen uptake [
m [0.11 m]; 83.1 kg [13.5 kg]; 4.03 L/min [0.51 L/min])
volunteered for this study, which was carried out over the
winter period (average daily temperature of 7?C). The
subjects had undertaken no formal acclimation or spent
any time in a hot environment over the preceding 2
months. The study was conducted in accordance with the
Declaration of Helsinki, was approved by the Local Ethics
Committee, and subjects provided written informed con-
]: 26 years [6 years]; 1.78
sent. Subjects visited the laboratory at a consistent time
of day for each visit.
A submaximal and a maximal exercise test were carried
out 4 days before the familiarization to determine the
workload required to elicit 38%
test involved four 5-minute bouts of exercise (49, 63, 77,
and 91 watts [W]) on an electronically braked cycle er-
gometer (Lode Excalibur, Groningen, The Netherlands)
with 5-minute rest between each workload. Pedal cadence
was maintained at 70 revolutions/min and expired gases
were measured continuously using an automated gas
analysis system (Oxycon Gamma, Mijnhardt, The Neth-
erlands). After a 15-minute rest, a continuous, incremen-
tal cycling test to volitional exhaustion was performed.
The initial workload was set at 70 W, with work rate in-
creasing by 35 W every 2 minutes until 6 minutes, and
every minute thereafter until exhaustion. Expired gases
were measured continuously to derive
. The submaximal
Subjects abstained from caffeine, tobacco, and alcohol in-
gestion and refrained from strenuous physical activity for
48 hours before testing. They recorded their dietary in-
take and physical activity for 48 hours before the famil-
iarization trial and were then asked to replicate these be-
fore the remaining trials. The evening before experimen-
tal testing, subjects consumed water at 10 mL/kg body
mass. Subjects consumed their last meal at least 6 hours
before testing, followed by a meal-replacement drink
(Complan, H.J. Heinz Co. Ltd., Hayes, UK) 2 hours before
reporting to the laboratory. The drink was reconstituted
by adding semiskimmed milk at 2.86 mL/kg body mass
to powder at 0.81 g/kg body mass. Constituents of the
drink based on the amount ingested by a 70-kg subject
were—energy: 1057 kJ; protein: 8.8 g; carbohydrates: 34.8
g (of which sugars: 26.3 g); fat: 8.5 g (of which saturates:
3.8 g); and sodium: 0.13 g. Along with the drink the sub-
jects consumed water at 10 mL/kg body mass minus the
volume of milk in the meal-replacement drink.
At least 4 days after the preliminary test and 1 week be-
fore the main trials, an initial trial was undertaken to
familiarize subjects with the investigative procedures,
verify , and establish sweat loss, which was to be re-
placed during the subsequent euhydrated exercise trials.
Because eHsp72 levels have been shown to fall back to
Cell Stress & Chaperones (2006) 11 (2), 129–134
The stress response during repeated exercise-heat exposures
Table 1 Urine specific gravity (USG) and nude body mass (NBM)
on arrival at the laboratory
Values are mean (SD).
resting levels 24 hours after exercise stress (Walsh et al
2001; Fehrenbach et al 2005), it was believed that even
after exercise combined with heat stress, 7 days would be
sufficient for levels to return to resting values with no
carryover from familiarization to main experimental trial.
On arrival at the laboratory subjects voided and eu-
hydration status was confirmed by the measurement of
urine specific gravity (USG ? 1.020, Multistix SG, Bayer
Diagnostics, Newburg, UK) (Armstrong et al 1994; Hoff-
man et al 1994). Nude body mass (NBM) was recorded
and a rectal thermistor was inserted. Subjects entered a
thermoneutral environment (mean [SD]; 26.7?C [0.5?C];
24.3% [4.0%] relative humidity [RH]) where they re-
mained seated for 30 minutes. Resting rectal temperature
(Tre) was recorded before entering the heat chamber
(mean [SD]; 38.0?C [0.1?C]; 60.0% [0.3%] RH; wind 1.18
m/s [0.02 m/s]), where exercise commenced immediate-
ly. This involved 2 hours of cycling at a workload initially
corresponding to 38%
on average, at 42.5% (3.6%) of
the 2 hours, because of
were given a 2-minute break every 30 minutes in order
to walk or stretch. On completion of exercise subjects
towel dried and NBM was remeasured, allowing the es-
timation of whole-body sweat loss.
, which equated to cycling,
over the course of
drift in the heat. Subjects
Subjects visited the laboratory on 3 consecutive days. The
first 2 visits (days 1 and 2) involved exercise-heat stress
and a resting blood sample was taken on the third visit.
The protocol and the environmental conditions were
identical to those of the familiarization trial. In addition,
before the 30-minute rest period in the thermoneutral en-
vironment, a cannula was inserted into an antecubital
vein while the subject was in the supine position. Arte-
rialized venous blood samples were taken pre-exercise
and immediately post- and 22 hours postexercise. The 22-
hour postexercise sample of day 2 was taken on the third
visit. Day 1’s 22-hour postexercise sample also served as
the resting sample on day 2. During exercise, sweat loss,
as calculated from the familiarization trial, was replaced
with a 20 mmol/L sodium chloride solution (Gisolfi et al
1990) every 15 minutes (Gisolfi and Duchman 1992). Pro-
vision was weighted toward the first hour of exercise;
20% (0 minutes), 20% (15 minutes), 15% (30 minutes),
15% (45 minutes), 15% (60 minutes), 5% (75 minutes), 5%
(90 minutes), and 5% (105 minutes). The initial 20% con-
sumed in the present study equated to 400–450 mL, after
which it was attempted to maintain this volume in the
stomach with regular fluid ingestion to promote gastric
emptying (Costill and Saltin 1974).
A rectal thermistor (Edale Instruments Ltd., Roedoure,
UK), inserted 10 cm beyond the anal sphincter, was used
for the measurement of Tre. Temperature was monitored
via a 1000 series 8-bit Squirrel data logger (Type 1002,
Grants, Cambridge, UK).
Blood sampling and analysis
Blood was collected from the indwelling catheter (Insyte
20 G, Becton Dickinson, Madrid, Spain) via vacutainers.
Hematocrit (Hct) was determined immediately from
whole-blood in triplicate, using the microcapillary tech-
nique. Serum was obtained from an SST vacutainer (BD
Vacutainer Systems, Plymouth, UK) by centrifugation and
frozen (?20?C) for later eHsp72 analysis. An enzyme-
linked immunosorbent assay was used to determine se-
rum Hsp72 concentration in duplicate, following the in-
structions of the manufacturer (StressGen Biotechnologies
[EKS-700], Victoria, BC, Canada).
Data are presented as mean (standard deviation). A 2-
factor (trial by time) repeated-measures analysis of vari-
ance (ANOVA) was performed to ascertain any treatment
or exercise effects on Treand eHsp72. A repeated-mea-
sures 1-way ANOVA was used to analyze resting eHsp72,
Hct, NBM, and USG to determine any change across the
3 days. The change in eHsp72 on both days was analyzed
by paired-sample Student’s t-test. Tukey’s honestly sig-
nificant difference post hoc analysis was used to locate
differences when the ANOVA revealed a significant dif-
ference. The significance level was set at P ? 0.05.
Subjects were euhydrated with no difference in NBM or
USG upon arrival at the laboratory on all 3 days (Table
Exercise in the heat resulted in an increased Treon both
days (day 1: rest 37.0?C [0.2?C], exercise 38.2?C [0.3?C];
day 2: rest 36.9?C [0.2?C], exercise 38.2?C [0.3?C]; P ?
Cell Stress & Chaperones (2006) 11 (2), 129–134
Marshall et al
[standard deviation]) during the initial stage of exercise-heat accli-
mation. ?, significant difference from rest; *, significant difference
from day 1 rest.
Extracellular heat shock protein 72 (eHsp72) levels (mean
72 (eHsp72) levels during 2 consecutive exercise-heat exposures.
Day 3 value is day 2’s 22-hour postexercise sample.
Individual subjects’ resting extracellular heat shock protein
0.001), with no difference in rest or exercise values be-
Exercise-heat stress increased Hct by the same amount
on both days (day 1: 1.7% [1.1%]; day 2: 1.7% [1.4%]).
Over the 3 days, resting Hct decreased by 5.0% (1.4%) (P
? 0.05) (day 1: 0.42 [0.03] ratio; day 3: 0.40 [0.02] ratio).
During exercise, eHsp72 increased by a similar amount
on both days (day 1: 1.26 ng/mL [0.80 ng/mL]; day 2:
1.29 ng/mL [1.60 ng/mL]). Resting eHsp72 levels de-
creased progressively in 6 of 7 subjects (Fig 1), becoming
significant (P ? 0.05) by day 3 (Fig 2).
In order to determine the effect of a single passive-heat
stress on eHsp72 in the experimental environment, a sub-
study was conducted on 5 subjects (mean [SD]; age,
height, and body mass: 25 years [5 years]; 1.85 m [0.11
m]; 79.2 kg [15.6 kg]). Two hours of passive heating (38.0 ?C
[0.1?C], 60.0% [0.2%] RH) was found to have no effect on
the eHsp72 concentration (baseline: 1.12 ng/mL [0.98 ng/
mL]; 2 hours of passive heating: 1.16 ng/mL [0.75 ng/
The effect of both acute and repeated exercise-heat stress
on human eHsp72 levels has been studied for the first
time. A reduced resting eHsp72 concentration was an ear-
ly adaptation to repeated exercise-heat exposures. The
stress imposed during each exercise-heat bout resulted in
an enhanced eHsp72 concentration; however, this eleva-
tion in eHsp72 was not altered by an identical exposure
24 hours previously.
The possibility that the decline in eHsp72 is due to
consumption of large volumes of fluid is unlikely because
the volume consumed was matched to the loss during
exercise, and resting USG did not differ between days.
However, resting Hct decreased (5%) after 2 repeated ex-
ercise-heat exposures because of a possible plasma vol-
ume expansion. No plasma volume data are available
from this study and comparison with the literature is dif-
ficult because plasma volume expansion is dependent on
both the sodium content of the diet (Armstrong et al
1993) and the fluid consumed during exercise (Costill et
al 1975). However, previous work in our laboratory found
a 6% expansion by the third day of exercise in humid
heat (H.C. Marshall and M.A. Nimmo, unpublished
data). The 36% reduction in eHsp72 over the same period
suggests that the plasma volume expansion was not the
sole factor determining the eHsp72 decrease. This reduc-
tion in eHsp72 was found despite an increased level in a
single outlying subject (Fig 1).
The concentration of eHsp72 is a result of the balance
between its release into, and removal from, the circula-
tion. A contribution to the reduction could have arisen
from a greater proportion being attached to the surface
of monocytes, eliciting an immunological or inflamma-
tory function (Asea et al 2000). A further function for
eHsp72, and a potential reason for its reduction, is that
cells less capable of synthesizing their own Hsp72, such
as neuroblastoma cells, can absorb and utilize Hsps in
their surrounding media (Guzhova et al 2001). A mech-
anism for receptor-facilitated transmembrane transport of
Hsp or Hsp-peptide complexes from the plasma mem-
brane into the cytosol has previously been suggested
(Castellino et al 2000; Basu et al 2001). During the initial
adaptation to exercise-heat stress, it is possible that cer-
tain cells are taking up eHsp72 to improve their stress
tolerance, without a concurrent increase in cellular re-
The reduction in resting eHsp72 levels, in addition to
the increased utilization described above, could be attri-
buted to a reduced intracellular production or a reduced
cellular release. However, this seems less likely because
Fehrenbach et al (2001) found human monocyte and
granulocyte Hsp72 protein levels to peak 24 hours after
an acute exercise-heat stress (60-minute run at 90% indi-
Cell Stress & Chaperones (2006) 11 (2), 129–134
The stress response during repeated exercise-heat exposures
vidual anaerobic threshold; 28?C, 50% RH) and remain
elevated above baseline 48 hours postexercise. Examina-
tion of these data suggests that the intracellular level of
Hsp72, at least in these cell types, is not a limiting factor
in its release into the circulation. In addition, an in vitro
study has indicated that the release of Hsp72 from epi-
thelial cells is manifested through a system of lipid rafts
and when heat-stimulated, translocation of Hsp70 to
these rafts is enhanced (Broquet et al 2003). More recent-
ly, it has been shown that the release of Hsp70 from pe-
ripheral blood mononuclear cells is not via a lipid-raft–
dependent pathway, but via exosomes, with an elevated
exosome Hsp70 content upon heat shock (Lancaster and
Febbraio 2005). Therefore, it appears that a reduced in-
tracellular production or cellular release are unlikely to
be responsible for the lowered resting eHsp72 levels in
the present study.
The enhanced eHsp72 concentration after exercise-heat
stress is supported by previous findings in less stressful
temperate environments (Walsh et al 2001; Lancaster et al
2004; Fehrenbach et al 2005). Although an enhanced
eHsp72 concentration was found after both stress expo-
sures, a blunted response during the second exposure
was not found, contrasting with the literature. This may
be due to the differing sample site (Mizzen and Welch
1988; Maloyan et al 1999), a lower initial heat stress (Miz-
zen and Welch 1988), or differing modes of heat accli-
mation (Maloyan et al 1999). Animal studies have sug-
gested that the release of endogenous Hsp72 during ex-
posure to stress is mediated in part by catecholamines
via stimulation of an ?1-adrenergic receptor–mediated
pathway (Fleshner et al 2004; Johnson et al 2005). Nor-
epinephrine is likely the primary catecholamine respon-
sible for Hsp72 release because, compared with epineph-
rine, it has a higher affinity for ?1-adrenergic receptors
(Pocock and Richards 2004) and the depletion of epi-
nephrine (95–99%) was found to have no effect on the
release of Hsp72 (Johnson et al 2005). Norepinephrine is
well known to increase during prolonged exercise in the
heat (Melin et al 1988; Hoffman et al 1994; Brenner et al
1997) and may be linked with the enhanced eHsp72 con-
centration during exercise-heat stress in the present
study. Acclimation has been found to have no effect on
norepinephrine at rest or during exercise (Febbraio et al
1994; Nielsen et al 1997), providing a possible reason for
the similar eHsp72 response during both days. However,
it suggests that the reduction in resting eHsp72 found in
the present study is due to factors other than norepi-
In conclusion, 2 consecutive exercise-heat exposures re-
sulted in a decreased resting concentration of eHsp72 in
humans. This was possibly due to an enhanced role in
the immune response or an enhanced cellular uptake by
cells unable to produce Hsps, providing an improved
stress tolerance in this initial, most stressful period of ac-
climation. The design of the experiment does not allow
us to determine whether the responsible factor for the
eHsp72 decline was heat, exercise, or a combination of
both. Further research is required to determine the exact
cause of the lowered resting eHsp72.
We are grateful to Glasgow City Council, Glasgow School
of Sport, and the West of Scotland Institute of Sport for
financial support for this study.
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