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Eur J Appl Physiol (2004) 91: 748–751

DOI 10.1007/s00421-004-1057-x

SH O RT CO MM U N IC A T IO N

Gavin Hayden Æ Helen C. Milne Æ Mark J. Patterson


Myra A. Nimmo

The reproducibility of closed-pouch sweat collection


and thermoregulatory responses to exercise–heat stress

Accepted: 15 January 2004 / Published online: 21 February 2004


 Springer-Verlag 2004

Abstract Seven active male subjects cycled for 60 min


at 29.5 (0.8)% peak work rate on three separate
Introduction
occasions in a hot environmental condition [36.0
The establishment of the reproducibility of a particular
(0.1)C, 60 (1)% relative humidity] in order to deter-
measure, whether physiological or measurement related,
mine the reproducibility of a closed-pouch sweat col-
is critical for research design to avoid occurrence of
lection technique for sweat composition at the scapula,
type II errors. The determination of sweat composition
forearm and thigh. To confirm that sweat composition
using a closed-pouch sweat collector (Brisson et al. 1991)
was not influenced by between-trial variations in
has been validated against other local sweat collection
sudomotor drive, local sweat rate, whole-body sweat
(Boisvert and Candas 1994) and whole-body wash-down
rate, heart rate (HR), rectal temperature (Tre) and
techniques (Patterson et al. 2000); however, the repro-
mean skin temperature (Tsk) responses were also
ducibility of this technique has not yet been established.
measured, consequently reproducibility was also
Shirreffs and Maughan (1997) established the reproduc-
established for these variables. Sweat composition did
ibility of a whole-body wash-down technique for deter-
not differ among trials, with the mean coefficients of
mination of sweat composition; however the reported
variation (CVs) for sweat [Na+], [K+] and pH being
coefficients of variation were high (15–23%) and were
10.4 (7.4)%, 8.1 (6.5)% and 1.3 (1.1)%, respectively.
likely to be related to differences in sweat rate between
Local sweat rates did not differ among the three trials
trials.
(P>0.05) although whole-body sweat rate was reduced
This project, therefore, was designed to determine the
in the third trial (P<0.05). The mean CVs were 11.0
reproducibility of the closed-pouch sweat collection
(7.8)% and 4.7 (1.6)% for local and whole-body sweat
technique described by Brisson et al. (1991) for sweat
rates, respectively. Between-trial differences were not
composition determination. Other physiological re-
evident for Tre, Tsk or HR with mean CVs of 0.3
sponses, including sweat rate, body temperature and heart
(0.2)%, 0.7 (0.6)% and 3.9 (1.7)%, respectively, al-
rate, were also monitored to confirm that the sudomotor
though HR tended to be greater in the first trial
drive was constant between trials. Consequently the
(P=0.08). It is proposed that moderate variations in
reproducibility of these variables was also established.
sweat composition were influenced by variations in the
local sweat rate, which were induced by application of
the pouch. Methods

Keywords Sweat collection Æ Thermoregulation Æ Subjects


Reproducibility Æ Exercise Æ Heat
Seven active males [age 21 (1) years; body mass 74.9 (6.2) kg; body
surface area 1.91 (0.10) m2; peak oxygen uptake (V_ O2peak) 3.80
(0.62) l min)1; mean (SD)] volunteered to participate in this
investigation. All procedures were approved by the University of
Strathclyde Ethics Committee, with written informed consent being
obtained from each subject.
G. Hayden Æ H. C. Milne Æ M. J. Patterson Æ M. A. Nimmo (&)
Department of Applied Physiology,
Faculty of Science, University of Strathclyde, Experimental design
76 Southbrae Drive, Glasgow, G13 1PP, UK
E-mail: m.a.nimmo@strath.ac.uk Each subject completed three submaximal cycling trials, 1 week
Tel.: +44-141-950-3176 apart, on a friction-braked bicycle ergometer (Monark, Sweden)
Fax: +44-141-950-3132 for 60 min at 29.5 (0.8)% aerobic work rate peak [W_ peak; 105
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(15) W] at 36.0 (0.1)C and 60 (1)% relative humidity. The load On completion of trials, subjects towel-dried themselves and
and the pedal cadence (70 rpm) were kept constant between tri- nude body mass was recorded. Whole-body sweat loss was deter-
als. Sweat from three skin areas was aspirated at the end of the mined by the change in body mass, uncorrected for respiratory
60-min trial, whereas heart rate (HR), and rectal (Tre) and skin water loss and metabolic water loss.
temperatures (Tsk) were recorded at 15-min intervals.
Workload corresponding to 30% W_ peak was calculated from a
continuous incremental cycling test to volitional exhaustion on an Statistical analysis
electronically braked cycle ergometer (Lode, The Netherlands).
Expired gases were measured to derive V_ O2peak using an automated To establish whether or not differences were evident between trials
gas analysis system (Oxycon Gamma, Mijnhardt, The Nether- for sweat rate and sweat composition a one-way analysis of vari-
lands). ance (ANOVA) was employed. For the repeated measures of HR,
The time of testing was consistent within a subject. In the 48-h Tre and Tsk, a repeated measures two-way ANOVA (time · trial)
period prior to each trial, subjects abstained from alcohol, caffeine was performed to establish any between-trial differences. A Tukey’s
and vigorous physical activity. Dietary intake, fluid consumption HSD post hoc analysis was employed to isolate any differences.
and physical activity were recorded 48 h prior to the first experi- The mean and SD were established for each subject, for each
mental trial and this was repeated for the subsequent trials. To variable, from the three trials, subsequently the coefficient of var-
ensure euhydration, subjects consumed 15 ml (kg body mass))1 of iation (100·SD/mean) was derived, i.e. a CV was established for
water 12 h before the trial and an additional 10 ml (kg body each subject for each variable. The CV data are expressed as means
mass))1 2 h before the trial. (SD) for the seven subjects. These results were then used for the
On arrival at the laboratory subjects voided and urine specific calculation of the mean CV at each time point. Data are presented
gravity was determined to ensure adequate hydration. Nude body as mean (SD) and a was set at the 0.05 level, unless otherwise
mass was recorded (plus or minus 2 g; Sartorius, Germany) by stated.
the investigator, after which the subject inserted a rectal therm-
istor (Edale Instruments, UK) and attached a HR monitor (Polar
Vantage, Finland) to their chest. Subjects then rested in a seated
position for 30 min in a thermoneutral room (25–27C) whilst Results
areas of skin on the right scapula, forearm and thigh were
cleaned with alcohol and deionised water prior to the attachment Sweat pH, [Na+] and [K+] exhibited no between-trial
of sweat patches. The sweat patch consisted of parafilm (24 cm2)
being attached to the skin with a wound dressing (Opsite, Smith
differences (Table 1). The CVs equated to absolute
and Nephew), as described by Brisson et al. (1991). Natural variations of 6.8–10.1 mmol l)1 for sweat [Na+], 0.4–
landmarks (i.e. freckles, moles etc.) were used to permit the exact 0.5 mmol l)1 for sweat [K+] and 0.08–0.10 for sweat
placement of the patch in subsequent trials. Skin thermistors pH.
(Edale Instruments) were placed adjacent to each sweat patch, Whole-body sweat rate was greater in trial 1 than in
and both Tre and Tsk were recorded via a 1,000 series 8-bit
Squirrel data logger (plus or minus 0.1C; Type 1002, Grants, trial 3 (P<0.05) although no between-trial differences
UK). Subjects then entered the environmental chamber and were observed for the regional sweat rates (Table 1). The
exercised for 60 min. exacerbated CV exhibited at the regional sites may
Weighted mean Tsk was calculated using: 0.42 scapula tem- explain the non-significant finding between days. That
perature + 0.19 forearm temperature + 0.39 thigh temperature
[modified from ISO 9886 (1992)]. Sweat was aspirated at the end of is, the absolute variation at the local skin regions
the 60-min trial and sweat rate was determined gravimetrically by (0.6–1.4 mg cm)2 min)1) was generally greater than the
the change in mass of the aspirating syringe (plus or minus 0.01 g; difference in whole-body sweat rate between trials 1 and
A&D Instruments, UK). Sweat [Na+] and [K+] were determined 3 (0.07 mg cm)2 min)1). Consequently the small change
by flame photometry (Sherwood, UK). Sweat pH was determined
with a micro-combination pH probe (Microelectronics, USA) after
in whole-body sweat rate could not be isolated by the
bubbling a 50-ll sample with 5% CO2. The intra-assay coefficients less sensitive closed-patch collection technique.
of variation (CVs) were 1.6%, 1.2% and 2.6% for [Na+], [K+] and Data on HR, Tre and Tsk were incomplete for one
pH, respectively. subject therefore these results are reported for six

Table 1 Sweat rate and


constituents, including Variable Trial 1 Trial 2 Trial 3 CV (%)
coefficients of variation (CV),
induced by 60 min exercise in Sweat rate (mg cm)2 min)1)
36C and 60% relative Whole body 0.85 (0.16) 0.82 (0.15) 0.78 (0.16)* 4.7 (1.6)
humidity on three separate Scapula 1.16 (0.60) 1.09 (0.49) 1.30 (0.79) 10.3 (5.2)
occasions. Values are means Forearm 1.00 (0.12) 1.04 (0.17) 0.98 (0.20) 6.3 (5.6)
(SD) Thigh 0.88 (0.35) 0.86 (0.32) 0.79 (0.38) 16.5 (9.1)
Sweat pH
Scapula 6.49 (0.32) 6.52 (0.35) 6.49 (0.32) 1.13 (0.49)
Forearm 6.48 (0.70) 6.42 (0.64) 6.37 (0.65) 1.23 (0.96)
Thigh 5.99 (0.73) 6.07 (0.74) 6.06 (0.75) 1.68 (1.56)
Sweat Na+ (mmol l)1)
Scapula 88.4 (30.4) 93.0 (25.1) 90.0 (22.1) 11.1 (10.6)
Forearm 83.1 (30.1) 79.5 (28.9) 79.9 (27.8) 8.4 (5.2)
Thigh 72.5 (19.9) 70.0 (26.7) 72.9 (23.1) 11.5 (6.3)
Sweat K+ (mmol l)1)
Scapula 4.18 (0.93) 4.34 (0.54) 4.32 (0.58) 8.9 (8.8)
Forearm 5.52 (0.65) 5.33 (0.63) 5.49 (0.95) 7.1 (4.8)
*Significantly different to trial 1 Thigh 5.85 (0.77) 5.34 (0.69) 5.34 (0.56) 8.4 (6.3)
(P<0.05)
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subjects. There was no difference in Tre [mean: 37.8 compliance, it cannot be definitively concluded that the
(0.1)C, 37.7 (0.2)C and 37.7 (0.2)C; P>0.05] or Tsk same Na+ intake was consumed. Stricter dietary control
[means: 36.7 (0.3), 36.7 (0.4) and 36.6 (0.5)C, P>0.05] could diminish the between-trial variation in sweat
between trials throughout the 60-min trial. The mean composition.
CVs were 0.3 (0.2)% and 0.7 (0.6)% for Tre and Tsk, The current reported sweat [Na+] CV was consider-
respectively, which equated to absolute variations of ably less than that reported by Shirreffs and Maughan
0.1C at 38.0C for Tre and 0.3C at 36.5C for Tsk. [1997; 17 (5%)] from a whole-body wash-down tech-
Mean exercise HR tended to be greater in trial 1 than nique. This larger CV was possibly due to the consid-
trials 2 and 3; however significance was not reached erable variation in sweat rate between trials, as it is well
[trial 1: 154 (10) beats min)1; trial 2: 148 (8) beats - established that sweat rate changes will alter sweat
min)1; trial 3: 148 (6) beats min)1; P=0.08]. The mean [Na+] (Allan and Wilson 1971). In the current investi-
CV was 3.9 (1.7)%, which equated to a variation of gation, local sweat rates were unaltered, therefore an
6 beats min)1 at 150 beats min)1. improved indication of between-trial biological variation
in sweat [Na+] was determined.
The low between-trial variation in rectal temperature
Discussion was expected and encouraging. To our knowledge this is
the first investigation that has quantified the reproduc-
We have established the reproducibility of the closed- ibility of Tre during exercise–heat stress in hot–humid
pouch sweat collection technique for the quantification conditions. While Barnett and Maughan (1993) reported
of sweat composition. The moderate between-trial var- an unaltered between-trial Tre response, no CV was
iation may have been affected by variations in the local reported.
sweat rates, which in turn were likely to be related to the While this is the first investigation to ascertain the
application of the pouch to the skin, since the variation reliability of HR during exercise in a hot environment,
in whole-body sweat rate seemed somewhat less than the CV observed was similar to that reported in cooler
that at the local skin regions. conditions (Wilmore et al. 1998). HR during submaxi-
The variation in sweat rate seemed to occur locally at mal exercise in the hot–humid conditions appeared
the sweat gland, since unaltered body temperatures and lower in trials 2 and 3 compared with that of trial 1,
a low variation in whole-body sweat rate suggest that the although significance was not reached (P=0.08). This
central sudomotor drive was equivalent between trials. finding could imply a heat acclimation effect; however,
Errors arising from the aspiration of the pouches seemed no other physiological variables exhibited responses
unlikely as 98.2 (0.6)% of a known volume was recov- conducive to heat acclimation; similarly Barnett and
ered during pilot testing. Further, the same experimenter Maughan (1993) reported no physiological refinements
aspirated all sweat pouches in the current study and the when exercise bouts were conducted on a weekly basis.
placement of the pouch was precisely controlled between The trend for a reduced exercising HR could be related
trials. to the familiarity of the subjects with the experimental
It is possible that alterations in the local environment protocol and procedures. Some subjects may have
induced by the sweat collector, including local skin experienced anxiety ensuing from unfamiliarity of exer-
temperature and hidromeiosis variations, were respon- cising in hot–humid conditions and the monitoring of
sible for the moderate between-trial sweat rate vari- physiological responses in a laboratory environment. If
ability. The skin temperature adjacent to the pouch anxiety levels were sufficient to augment sympathetic
confirmed no gross change in skin temperature of the nervous activity the greater HR response in the first
body segment; however, the temperature variation under trial could be explained. If an exacerbated sympathetic
the pouch was not determined since this could have nervous activity was evident in the first trial this may
introduced contamination and/or compromised the seal explain the reduced whole-body sweat rate between
of the pouch. Further it is unlikely that local skin tem- trials 1 and 3, considering the non-thermal drive for
perature would be measured when employing the sweat sweating has been linked to greater sympathetic nervous
collection technique in future investigations. Regardless activity (Shibasaki et al. 2001). Therefore future inves-
of the cause of the varied sweat rate and subsequent tigators should consider the implementation of an initial
sweat composition when employing the current tech- trial to familiarise subjects with the experimental pro-
niques, the treatment effects needed to ensure the tocol and procedures to alleviate possible differences in
avoidance of a type II error have been clearly estab- sympathetic nervous activity between treatment trials.
lished. This study established the reproducibility of the
The moderate variation in sweat [Na+] could also closed-pouch sweat collection technique for determina-
have been related to between-trial variation in dietary tion of sweat composition during exercise-heat stress. It
Na+ intake. Dietary Na+ intake has been reported to is proposed that moderate variations in sweat compo-
affect sweat [Na+] during heat acclimation (Armstrong sition were influenced by similar variations in the local
et al. 1985). While subjects were instructed to consume sweat rate and were induced by application of the
the same foods in the 48-h period before a trial, com- pouch. The whole-body sweat rate, Tre, Tsk and HR
plete food diaries and were questioned in regard to exhibited, as expected, low CVs in the hot–humid
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conditions. However, the reduction in whole-body sweat Brisson GR, Boisvert P, Peronnet F, Perrault H, Boisvert D,
rate between trials 1 and 3, combined with the tendency Lafond JS (1991) A simple and disposable sweat collector. Eur J
Appl Physiol 63:269–272
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protocol and procedures before conducting experimental Switzerland
trials when exercising in hot–humid conditions. Patterson MJ, Galloway SDR, Nimmo MA (2000) Variations in
regional sweat composition in normal human males. Exp
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Shibasaki M, Kondo N, Crandall CG (2001) Evidence for metab-
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