Effects of Prolonged Exercise in the Heat and Cool Environments on Salivary Immunoglobulin A among Recreational Athletes

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IOSR Journal of Sports and Physical Education (IOSR-JSPE)
e-ISSN: 2347-6737, p-ISSN: 2347-6745, Volume 3, Issue 4 (Jul. – Aug. 2016), PP 51-56
www.iosrjournals.org
DOI: 10.9790/6737-03045156 www.iosrjournals.org 51 | Page
Effects of Prolonged Exercise in the Heat and Cool Environments
on Salivary Immunoglobulin A among Recreational Athletes
Ayu S. Muhamad1, Chee K. Chen2, Ayunizma Ayub3, Nur S. Ibrahim4
1(Exercise and Sports Science Programme, School of Health Sciences, Health Campus, Universiti Sains
Malaysia, Malaysia)
Abstract: This study aims to determine the effects of prolonged exercise in the heat (31°C) and cool (18°C)
environments on salivary Immunoglobulin A (SIgA) among recreational athletes. Thirteen healthy male
participants (age: 20.9 ± 1.3 years old) were recruited and randomised in this cross-over study. In this study,
participants performed the exercise trials in the heat environment at 31°C first followed by another exercise
trial in the cool environment trial at 18°C or vice versa with one week of recovery period. Physiological
parameters (heart rate, body weight changes and oxygen uptake) as well as room temperature and relative
humidity were recorded. Cool water (3 ml.kg-1 body weight) was given to the participants at every 20 min
during both exercise trials. Saliva samples were collected to calculate the saliva flow rate and analysed for
salivary Immunoglobulin A (SIgA) concentrations and secretion rate. Paired t-test and two-way ANOVA with
repeated measures were performed to analyse the data. The results revealed that saliva flow rate, SIgA
concentration, and SIgA secretion rate did not significantly different between exercise trial in the heat and in
the cool environments. However, prolonged exercise significantly decreased (p < 0.05) saliva flow rate in both
trials with the values return to baseline 1 h post exercise. Salivary IgA concentration and secretion rate were
not affected by prolonged running. As a conclusion, SIgA responses did not affected by ambient/room
temperature. In addition, prolonged exercise with adequate fluid intake during exercise did not supress SIgA
responses thus may not increase infection risk among athletes.
Keywords: exercise, immune function, mucosal immunity, salivary antimicrobial protein
I. Introduction
Numerous studies have been conducted to investigate the effects of exercise on immune function. In
general, it has been demonstrated that a regular moderate intensity exercise improves immune function while
prolonged high intensity exercise may suppress immune function [1]. Therefore, athletes and fitness enthusiasts
are very concerned about the effects of exercise on the body immune function. This is because poor health status
can eventually leads to a high risk of getting infection, especially on the upper respiratory tract infection (URTI)
[2, 3]. The suppression of the immune function may in turn affect the sports performance of the athletes
particularly during training and competition. The depression of immune function is most pronounced when the
exercise is continuous, prolonged (> 1.5 h), in a moderate to high intensity (55 – 75% of VO2max) [4]. However,
the suppression of immune function may return back to the baseline value within hours depending on the
exercise intensity and duration [5].
With regards to environmental temperature, usually, it has been found that exercise in extreme
temperature may negatively affect the immune function compared to exercise in the thermoneutral and cool
condition [6, 7]. Hence, exercising in different environment has different effects on the body immune response.
Nowadays, most athletes travel from one county to another country to participate in different type of
competition. Even in stage world sporting events such as the Olympic Games, an athlete are required to compete
in adverse environmental conditions, for instance in the extreme heat and humidity of Athens in 2004.
Therefore, it is crucial to measure the SIgA responses because it is one of the antimicrobial proteins
(AMPs) which act as the body’s first line of defence [8]. It involved in the immune exclusion by preventing
antigens and microbes from adhering to and penetrating the epithelium, intracellular neutralization, and immune
excretion by binding to antigens in the lamina propria [9]. Besides, numerous studies have observed a decline in
SIgA levels after prolonged intense exercise in endurance athletes, and sometimes associated with an increased
incidence of URTI symptoms [8]. Nevertheless, even though findings regarding the effects of exercise on SIgA
level are numerous, but the findings are contradictory [10, 11]. In addition, studies on SIgA in saliva with
regards to exercise in the heat and cool environment are also still tremendously limited.
Therefore, this study is warranted to investigate the effects of prolonged running in the heat and cold
temperature on SIgA responses among recreational athletes. Most of the previous studies were carried out in
room temperature, thus this study will provide useful knowledge regarding the effects of exercise in heat and
cool environments on SIgA.

Effects of Prolonged Exercise in the Heat and Cool Environments on Salivary Immunoglobulin A
DOI: 10.9790/6737-03045156 www.iosrjournals.org 52 | Page
II. Methodology
Research Design
A randomised, cross-over trial was employed for the present study. Participants performed 2 separate
trials; exercise in the heat followed by exercise in the cool environment or vice versa. Recovery period between
these two trials was one week. All of the tests were conducted in the laboratory of Sports Science Unit (SSU),
Universiti Sains Malaysia (USM).
Participants and Sample Size Calculation
Participation in this study was in voluntary basis. This study has been approved by the Human
Research and Ethics Committee, Health Campus, Universiti Sains Malaysia, Kelantan (USM/JEPeM/140361).
In this study, opportunistic or convenience sampling was used whereby 13 active recreational athletes were
recruited among USM students. Participants were healthy males, aged between 18 and 30 years old, non-
smokers, and exercise regularly (at least 3 times per week with at least 30 min per session). Those who were
having cold or respiratory tract infection at least 2 weeks prior to the study and on medication were excluded in
this study. Throughout the study period, participants were required to abstain from taking any supplements that
are known to affect immune function, e.g. probiotics, vitamin C, vitamin D and plant polyphenols like
Quercetin.
Exercise Trials Procedures
During the first 3 visits to the laboratory, participants performed three preliminary tests which include
sub-maximal test, maximal oxygen uptake (VO2max; modified Astrand protocol4) test, and familiarisation trial.
The preliminary tests were carried out on a motorised treadmill (TrackMaster TMX425CP, USA) to determine
participant’s VO2max, to calculate each participant’s speed at 60% VO2max, and to familiarise them with the
running trial protocol. The 4th and 5th visits to the laboratory were for carried out the actual running trial;
running for 90 min at 60% of their respective VO2max. Participants performed 2 running trials in 2 different
environments; heat (31°C) and cool (18°C) environments. The order of the running trials was randomised. Heat
environment was maintained at 31°C by using halogen lamps (Philips-500W, France) whereby cool
environment was set at 18°C by adjusting the temperature on the air conditioner (York, USA). The relative
humidity in both running trials was maintained at 70% by using a heated water-bath (Memment W350t,
Germany).During each running trial, participant came to the laboratory in the morning after an overnight fast.
Upon arrival, participants were asked to measure their nude body by using a body composition analyser (TBF-
410 Tanita, Japan) in a closed room. Following that their saliva sample (2 mL) was collected by 5 min un-
stimulated dribbling into a pre-weighed sterile bijou tube (Sterilin, Staffordshire, UK). They were asked to sit on
a chair, lean the head forward and let the saliva passively dribble into the tube; without using their tongue or any
mouth movement. Following that, the bijou tube with saliva was weighed and recorded. Then, participants were
cannulated for blood drawing purposes. Blood sample (5 mL) was collected into a K3EDTA collection tube
(Sekusui Insepack, Japan). Patency of the cannula was maintained by heparinised saline whereby 0.2 ml of
heparinised saline was injected into the extension tube after each blood withdrawal to avoid blood clotting. After
that, participants were given a standardised breakfast; 2 pieces of white bread (Gardenia®, Malaysia) and 250
ml of cool plain water. After resting for half an hour the running trial was begun.
The running trial was begun with a 5 min warm-up at 50% of participant’s respective VO2max followed
by 90 min running trial at 60% of participant’s respective VO2max. The heart rate (heart rate sensor: Sport Tester
PE3000, Polar, Finland), oxygen uptake (pre-calibrated gas analyser system: VMax-SensorMedics, USA), room
temperature and relative humidity (psychrometer: Extech Instruments RH305, USA) were measured before
warm-up, after warm-up, at every 20 min during the running trial and at the end of the trial. During the 90 min
of the running trial, participants were asked to drink 3 mL.kg-1 body weight of cool water at every 20 min to
avoid any adverse effects of dehydration. In addition, participant was directed with a standing fan with speed
level 1 to mimic air flow in an open environment throughout the running trial. The second saliva sample was
collected immediately at the end of the trial while the final saliva samples were collected 1 h post exercise.
During this 1 h period, participants were resting in a comfortable room. Saliva samples were analysed for SIgA
concentrations by using a commercially available reagent kit (LDN Labor Diagnostica Nord GmbH & Co. KG,
Germany) via an ELISA (Enzyme-Linked Immunosorbent Assay) method. The calculations for determining
saliva volume/weight, flow rate, and saliva antimicrobial proteins’ secretion rate (SIgA and α-amylase) in this
study are as follow:
 Saliva volume (ml) = Difference in weight (g) of bijou tube after collection of saliva assuming a saliva
density of 1.0 g/ml
 Saliva flow rate (ml/min) = Saliva volume(ml)/ Collection time (min)
 SIgA secretion rate (µg/min) = Saliva flow rate (ml/min) × Saliva antimicrobial protein concentration
(µg/ml)

Effects of Prolonged Exercise in the Heat and Cool Environments on Salivary Immunoglobulin A
DOI: 10.9790/6737-03045156 www.iosrjournals.org 53 | Page
Statistical Analysis
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 22.
Descriptive statistics were performed on physiological characteristics. Room temperature, relative humidity,
body weight changes were analysed by paired t-test. Two-way ANOVA with repeated measures was performed
to measure significant differences between trials and within trials for lysozyme responses, oxygen uptake, heart
rate, and rate of perceived exertion. The accepted level of significance is set at p < 0.05. Results were reported
as means ± standard deviation (SD).
III. Results
Mean body composition and cardiorespiratory fitness of the participants were presented in Table 1.
Whereas, room temperature, relative humidity and body weight changes of the participants were presented in
Table 2.
Table 1: Body composition and cardiorespiratory fitness of the participants
Variable (N=13)
Body Composition
Age (years)
20.9 ± 1.3
Weight (kg)
63.2 ± 7.8
Height (cm)
167.6 ± 5.0
BMI (kg.m-2)
22.4 ± 2.1
Cardiorespiratory Fitness
VO2max (mL.kg-1.min-1)
47.0 ± 4.1
VO2 at 60% VO2max (mL.kg-1.min-1)
32.3 ± 3.4
Values are mean ± SD
Table 2: Room temperature, humidity and body weight changes of the participants
Variable (N=13)
Heat Trial
Cool Trial
Room Temperature (ºC)
31.0 ± 0.2
18.2 ± 0.3 *
Relative Humidity (%)
70.3 ± 1.0
70.8 ± 1.0
Pre-exercise body weight (kg)
63.2 ± 7.8
63.3 ± 7.9
Post-exercise body weight (kg)
62.3 ± 7.9
62.8 ± 8.0
Body weight changes (%)
1.5 ± 0.6
0.7 ± 0.5 *
Values are mean ± SD.
*significantly different from the heat trial (p < 0.05)
Heart rate was significantly increased over time (p < 0.001) in both trials. However, it was significantly
higher (p < 0.001) in the heat trial compared to cool trial. Post-exercise heart rate of the participants for heat and
cool trials was 167.2 ± 12.1 and 142 ± 3.3 beats.min-1 respectively. Similarly, the oxygen uptake during exercise
was significantly increased (p < 0.001) from baseline value until end of warm-up session at approximately 50%
VO2max, but then it was relatively stable throughout exercise at approximately 60% VO2max with a significant
difference (p = 0.027) found between trials. There was a significant main effect of time on saliva flow rate
during both trials (p = 0.025) (Fig. 1) whereby it was significantly decreased post-exercise. Nevertheless, it was
increased to approximately baseline values at 1 h post-exercise in both trials. However, there was no significant
difference (p > 0.05) on saliva flow rate between trials. Besides, there were also no significant main effects of
time (p > 0.05) on SIgA concentration (Fig. 2) and secretion rate (Fig. 3) in both trials. In addition, there was no
significant difference (p > 0.05) on SIgA concentration and secretion rate between trials.
++ significantly different from respective resting value (p < 0.01)
Figure 1: Saliva flow rate (mL.min-1) in the heat and cool trials

Effects of Prolonged Exercise in the Heat and Cool Environments on Salivary Immunoglobulin A
DOI: 10.9790/6737-03045156 www.iosrjournals.org 54 | Page
Figure 2: SIgA concentrations (µg.mL-1) in the heat and cool trials.
+ Significantly different from respective resting value (p < 0.05).
Figure 3: SIgA secretion rate (µg.min-1) in the heat and cool trials.
IV. Discussion
The main finding in this study is to investigate the effects of prolonged running in the heat and cool
temperature on SIgA responses among recreational athletes. In the present study, SIgA concentration (Fig. 2)
and secretion rate (Fig. 3) were not significantly different during exercise between the heat and cool trials which
is in agreement with the previous studies [12, 13]. However, the previous and the present study also found that
within each trial, the prolonged exercise was significantly decreased only in SIgA secretion rate (Fig. 3) [14, 15]
which was contrary to the SIgA concentration (Fig. 2). Nevertheless, there were also contradicting study which
reported that cold temperature has been associated with the increased [16], no changed [17] and decreased [18,
19] in SIgA responses. Other investigators also reported there were actually a significant increase [20, 21] in
SIgA after exercise.
Theoretically, SIgA is secreted by both acinar and ductal units under the stimulation of α - and β-
adrenergic and peptidergic receptors where, stimulation of β-adrenoreceptors increased SIgA secretion rate.
However, prolonged β-adrenoreceptors stimulation appeared to reduce the replenishment of SIgA into the
glandular pool [22]. The inconsistency of the secretory immune response of SIgA concentration and secretion
rate may be attributed to the interaction between different types of stimulation and their receptors during

Effects of Prolonged Exercise in the Heat and Cool Environments on Salivary Immunoglobulin A
DOI: 10.9790/6737-03045156 www.iosrjournals.org 55 | Page
exercise. This discrepancy may also be attributed to the differences during the time of the saliva collection [23]
and due to the different methods of expressing SIgA, nutritional status of the individual, and the exercise
protocol employed.
Moreover, the saliva flow rate during exercise in the heat and cool trials was not significantly
difference (Fig. 1) and was similar to the previous study [24-26]. This was associated with the stimulates
sympathetic nervous system due to performing exercise [27]. Therefore, it was suggested that sympathetic
nervous system activity influenced the decreased of saliva flow rate [28]. Hence, this explains the reduction on
the saliva flow rate found in the present study.
Overall, participants recruited was within the range of Asian populations’ BMI [29] which were not
obese and was considered ‘average’ in term of cardiorespiratory fitness [30] (Table 1). While, the room
temperature of 31°C and relative humidity of 70% were selected in the present study based on the numerous
studies conducted in the heat had set the room temperature and relative humidity at about these values [31-33].
This is also similar to the cool trial which 18°C was selected as the temperature for the cool environment [34-
36] (Table 2). Besides, the protocol was chosen as it was intended to suppress the immune function temporarily
in order to determine the effects of heat and cool environments on the immune function [1]. This is due to the
depression of immune function is most prominent when exercising in continuous and prolonged (> 1.5 h) at
moderate to high intensity. The VO2 values (Table 2) were also being measured to avoid bias between trials.
Therefore, based on the present study, both VO2 values were found with no significantly difference. Hence, the
participant performed the exercise at the same intensity as suggested by previous study in both of the trials.
Furthermore, heart rate in the present study was significantly increased during exercise and higher
when exercising in a hot temperature compared to cool environment (Table 2)which is consistence with the
previous study [6, 36]. Other than that, the body weight changes (Table 2) were also found higher in during the
heat compared to the cool trial. Thus, based on the present study, it was found that there were significantly
difference between heat and cool trial and it was predictable as in the previous finding [6]. The measurement of
body weight changes was crucial because performance can actually alter when dehydration exceeds 2% of body
mass [37, 38]. In the present study, the amount of fluid given during both trials was 3mL.kg-1 body weight at
every 20 min. This amount of fluid has been used in previous studies to rehydrate the participants during
exercise longer than 1 h [31-33]. Since the body weight changes in both trials were less than 2%, it is considered
that the amount of fluid ingested was sufficient to avoid the adverse effects of dehydration in this study.
V. Conclusion
The present study showed that room or ambient temperature (31°C vs 18°C) does not affect the
changes in SIgA responses during prolonged exercise among recreational athletes. However, several
physiological parameters did affected by the heat temperature. Thus, fluid intake during exercise especially
while performing exercise in the heat is warranted. Future studies should measure other parameters which
include other salivary antimicrobial proteins to clearly understand their responses in different temperatures.
Acknowledgements
The authors would like to thank the participants and laboratory staff of Sports Science Laboratory,
USM. This research was financially supported by a USM short-term grant (304/PPSP/61313029).
References
[1]. Gleeson M. Immune system adaptation in elite athletes. Current opinion in clinical nutrition and metabolic care. Nov
2006;9(6):659-665.
[2]. Gleeson M, Bishop NC, Stensel DJ, Lindley MR, Mastana SS, Nimmo MA. The anti-inflammatory effects of exercise: mechanisms
and implications for the prevention and treatment of disease. Nature reviews. Immunology. Sep 2011;11(9):607-615.
[3]. Walsh NP, Gleeson M, Shephard RJ, et al. Position statement. Part one: Immune function and exercise. Exercise immunology
review. 2011;17:6-63.
[4]. Gleeson M. Can Nutrition Limit Exercise-Induced Immunodepression? Nutrition Reviews. 2006-03-01 00:00:00 2006;64(3):119-
131.
[5]. Gleeson M. Immune Function and Exercise. European journal of sport science. 2005;4(3):15.
[6]. Galloway SD, Maughan RJ. Effects of ambient temperature on the capacity to perform prolonged cycle exercise in man. Medicine
and science in sports and exercise. Sep 1997;29(9):1240-1249.
[7]. Niess AM, Fehrenbach E, Lehmann R, et al. Impact of elevated ambient temperatures on the acute immune response to intensive
endurance exercise. European journal of applied physiology. May 2003;89(3-4):344-351.
[8]. Gleeson M, Pyne DB. Special feature for the Olympics: effects of exercise on the immune system: exercise effects on mucosal
immunity. Immunology and cell biology. Oct 2000;78(5):536-544.
[9]. Lamm ME. Current concepts in mucosal immunity. IV. How epithelial transport of IgA antibodies relates to host defense. Am. J.
Physiol. 1998;274((4 PT 1)):3.
[10]. Fricker PA, Pyne DB, Saunders PU, Cox AJ, Gleeson M, Telford RD. Influence of training loads on patterns of illness in elite
distance runners. Clinical journal of sport medicine : official journal of the Canadian Academy of Sport Medicine. Jul
2005;15(4):246-252.

Effects of Prolonged Exercise in the Heat and Cool Environments on Salivary Immunoglobulin A
DOI: 10.9790/6737-03045156 www.iosrjournals.org 56 | Page
[11]. Tiollier E, Chennaoui M, Gomez-Merino D, Drogou C, Filaire E, Guezennec CY. Effect of a probiotics supplementation on
respiratory infections and immune and hormonal parameters during intense military training. Military medicine. Sep
2007;172(9):1006-1011.
[12]. Laing SJ, Blackwell J, Gwynne D, Walters R, Walsh NP. Neutrophil degranulation response to 2 hours of exercise in a 30 degrees
C environment. Aviation, space, and environmental medicine. Nov 2005;76(11):1068-1073.
[13]. Akimoto T, Kim K, Yamauchi R, et al. Exercise in, and adaptations to a cold environment have no effect on SIgA. The Journal of
sports medicine and physical fitness. Sep 2009;49(3):315-319.
[14]. Gleeson M, McDonald WA, Pyne DB, et al. Salivary IgA levels and infection risk in elite swimmers. Medicine and science in
sports and exercise. Jan 1999;31(1):67-73.
[15]. Nieman DC. Is infection risk linked to exercise workload? Medicine and science in sports and exercise. Jul 2000;32(7 Suppl):S406-
411.
[16]. Willemsen G, Ring C, Carroll D, Evans P, Clow A, Hucklebridge F. Secretory immunoglobulin A and cardiovascular reactions to
mental arithmetic and cold pressor. Psychophysiology. May 1998;35(3):252-259.
[17]. Winzer A, Ring C, Carroll D, Willemsen G, Drayson M, Kendall M. Secretory immunoglobulin A and cardiovascular reactions to
mental arithmetic, cold pressor, and exercise: effects of beta-adrenergic blockade. Psychophysiology. Sep 1999;36(5):591-601.
[18]. Ring C, Harrison LK, Winzer A, Carroll D, Drayson M, Kendall M. Secretory immunoglobulin A and cardiovascular reactions to
mental arithmetic, cold pressor, and exercise: effects of alpha-adrenergic blockade. Psychophysiology. Sep 2000;37(5):634-643.
[19]. Willemsen G, Carroll D, Ring C, Drayson M. Cellular and mucosal immune reactions to mental and cold stress: associations with
gender and cardiovascular reactivity. Psychophysiology. Mar 2002;39(2):222-228.
[20]. Sari-Sarraf V, Doran DA, Clarke ND, Atkinson G, Reilly T. Effects of carbohydrate beverage ingestion on the salivary IgA
response to intermittent exercise in the heat. International journal of sports medicine. Sep 2011;32(9):659-665.
[21]. Blannin AK, Robson PJ, Walsh NP, Clark AM, Glennon L, Gleeson M. The effect of exercising to exhaustion at different
intensities on saliva immunoglobulin A, protein and electrolyte secretion. International journal of sports medicine. Nov
1998;19(8):547-552.
[22]. Proctor GB, Garrett JR, Carpenter GH, Ebersole LE. Salivary secretion of immunoglobulin A by submandibular glands in response
to autonomimetic infusions in anaesthetised rats. Journal of neuroimmunology. Mar 2003;136(1-2):17-24.
[23]. Burns VE, Ring C, Harrison LK, Carroll D, Drayson M. Reductions in secretory immunoglobulin A to cold pressor stress are not
influenced by timing of saliva sampling. Biological psychology. Mar 2004;66(1):91-98.
[24]. Tharp GD, Barnes MW. Reduction of saliva immunoglobulin levels by swim training. European journal of applied physiology and
occupational physiology. 1990;60(1):61-64.
[25]. Nieman DC, Nehlsen-Cannarella SL, Markoff PA, et al. The effects of moderate exercise training on natural killer cells and acute
upper respiratory tract infections. International journal of sports medicine. Dec 1990;11(6):467-473.
[26]. Nieman DC, Henson DA, Fagoaga OR, et al. Change in salivary IgA following a competitive marathon race. International journal
of sports medicine. Jan 2002;23(1):69-75.
[27]. Mueller PJ. Exercise training and sympathetic nervous system activity: evidence for physical activity dependent neural plasticity.
Clinical and experimental pharmacology & physiology. Apr 2007;34(4):377-384.
[28]. Ford J TN, Dix CA, Fallowfield JL. The effect of fluid replacement and heat on salivary flow rate and optical density at 280 nm in
response to exercise. J. Sports Sci. 1997;15:5.
[29]. Choo V. WHO reassesses appropriate body-mass index for Asian populations. Lancet. Jul 20 2002;360(9328):235.
[30]. McArdle WD, Katch FI, Katch VL. Exercise Physiology: Energy, Nutrition, and Human Performance: Lippincott Williams &
Wilkins; 2001.
[31]. Muhamad AS, Chen, C. K., Ooi, F. K., Abdullah, M. R., & Lam, C. K. . Effects of Eurycoma longifolia Jack Supplementation on
Recreational Athletes' Endurance Running Capacity and Physiological Responses in the Heat. International Journal of Applied
Sports Sciences. 2010;22(2):19.
[32]. Keong CC, Singh HJ, Singh R. Effects of palm vitamin e supplementation on exercise-induced oxidative stress and endurance
performance in the heat. Journal of sports science & medicine. 2006;5(4):629-639.
[33]. Saat M, Tochihara Y, Hashiguchi N, Sirisinghe RG, Fujita M, Chou CM. Effects of exercise in the heat on thermoregulation of
Japanese and Malaysian males. Journal of physiological anthropology and applied human science. Jul 2005;24(4):267-275.
[34]. Gueritee J, Tipton MJ. The relationship between radiant heat, air temperature and thermal comfort at rest and exercise. Physiology
& behavior. Feb 2015;139:378-385.
[35]. Stensrud T, Stang J, Thorsen E, Braten V. Exhaled nitric oxide concentration in the period of 60 min after submaximal exercise in
the cold. Clinical physiology and functional imaging. Mar 2016;36(2):85-91.
[36]. Peake J, Peiffer JJ, Abbiss CR, Nosaka K, Laursen PB, Suzuki K. Carbohydrate gel ingestion and immunoendocrine responses to
cycling in temperate and hot conditions. International journal of sport nutrition and exercise metabolism. Jun 2008;18(3):229-246.
[37]. Sawka MN, Coyle EF. Influence of body water and blood volume on thermoregulation and exercise performance in the heat.
Exercise and sport sciences reviews. 1999;27:167-218.
[38]. Cheuvront SN, Montain SJ, Sawka MN. Fluid replacement and performance during the marathon. Sports medicine. 2007;37(4-
5):353-357.