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Department of Sport, School of Physical Education and Sport, University of Sao Paulo, Sao Paulo, Brazil; 2Department of
Physical Education, State University of Londrina, Parana, Brazil; 3Department of Sport, Integrated Support Center for
AthletesPinheiros Sport Club, Sao Paulo, Brazil; and 4Department of Sport, School of Arts, Sciences and Humanities,
University of Sao Paulo, Sao Paulo, Brazil

ABSTRACT the likelihood to decrements on mucosal immunity and its

Moreira, A, Freitas, CG, Nakamura, FY, Drago, G, Drago, M, consequent risk to upper respiratory tract infections, which in
and Aoki, MS. Effect of match importance on salivary cortisol turn might affect the performance.
and immunoglobulin A responses in elite young volleyball KEY WORDS salivary steroids, stress, mucosal immunity, team
players. J Strength Cond Res 27(1): 202207, 2013The sports, monitoring training
purpose of this study was to compare the session ratings of
perceived exertion (Session-RPE) responses and the salivary
cortisol (sC) and immunoglobulin A (SIgA) levels between

a regular season match (RM) and the final championship match sychosocial stress leads to the activation of the
hypothalamus-pituitary-adrenal axis in humans
(FM) in elite male volleyball players against the same opponent
(12). From a psychobiological perspective, stress
team. Higher importance was assumed for FM because this
is defined as a particular relationship between the
match would define the championship team. Session-RPE was
person and the environment that is appraised by the person
obtained after 30 minutes of each match using the CR-10 as taxing or exceeding his or her resources and endangering
scale. Saliva samples were collected before and after each his or her well-being (17). Considering this definition, Gaab
match and during a rest day (baseline) at the same period of the et al. (12) suggested that stress in a given situation should be
matches. The SIgA and sC concentrations were measured by viewed as the result of a cognitive appraisal process, inducing
enzyme-linked immunosorbent assay. Greater Session-RPE a stress response related to emotional, physiological, and
was observed for FM as compared with RM (p , 0.01). The behavioral dimensions. Dhabhar and McEwen (7) extended
analysis of variance showed greater sC concentrations to FM the stress concept providing an integrated definition: stress
as compared with RM for both prevalues and postvalues and is a constellation of events, consisting of a stimulus (stressor)
compared with baseline (p , 0.05). Significant lower SIgA that precipitates a reaction in the brain (stress perception),
that activates physiological fight-or-flight systems in the
prevalues were noted for FM. In conclusion, the results showed
body (stress response).
that match intensity, cortisol concentration, and SIgA prelevel
Thus, it could be argued that with the aim to investigate
were affected by the match importance. These results indicate
the stress response within different settings, an integrated
that monitoring session-RPE, sC, and SIgA responses, in approach that takes into account some specific perceptual
conjunction, during training and competition, would provide and biological indicators is desirable. In a sports setting,
valuable informations regarding how athletes cope with sports athletes are constantly submitted to a wide range of stressors,
induced stress. This study provided knowledge about the effect in both physiological and psychological contexts (26). For
of match importance on salivary markers related to stress that instance, the daily training load can be judged as the main
may help coaches to avoid excessive training loads reducing source of physiological stress. In addition, many aspects of
sports official competitions such as the pressure to achieve
optimal outcomes, the unpredictable environment related to
Address correspondence to Alexandre Moreira, official matches, the importance of the match, among others,
27(1)/202207 emerge as potent psychological stressors.
Journal of Strength and Conditioning Research A reliable and practical method to assess internal training
2013 National Strength and Conditioning Association load based on session ratings of perceived exertion (Session-
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RPE) has been proposed by Foster (11). The Session-RPE METHODS

method has been extensively used to assess physical effort Experimental Approach to the Problem
intensity in many different conditions (1,14,1820). Because The study group was evaluated across 2 official matches
Session-RPE also reflects the match intensity (5,11,14), it is over 3 weeks, during the competitive volleyball season (fall
reasonable to expect that as competition progresses with season). Both official matches were played between approx-
more difficult games and opponents, session-RPE score
imately 18:30 and 20:30 hours. The opposing team was the
would accordingly increase.
same in both occasions. The opposing and the investigated
The impact of the exercise and sports-related stress on teams reached the first and second position, respectively, at
hormonal responses has been previously demonstrated the main, State, Brazilian under-19 volleyball championship.
(2,8,13,25,27). In fact, many investigations focusing on real Some of the investigated and opposing athletes were
sports competitions highlighted that these situations elicit members of the under-19 national Brazilian team during
additional psychological stressors along with the physi- the period of the investigation. The investigated team was
cal demands, maximizing stress hormone responses (e.g., defeated on both occasions (3 vs. 0). The first official match
cortisol) (8,10,13,25). Because competition can be charac-
belonged to the regular season phase (qualifying; RM) and the
terized as a situation associated with ego involvement,
second official match was the final championship match
novelty, anxiety, lack of control, and unpredictability, which (FM). Each game was preceded by a 30-minute warm-up of
in turn, might lead to negative affective properties (4), it light aerobic exercise, volleyball drills, and dynamic stretching
seems reasonable to expect greater hormonal responses to of the major muscle groups. All of the investigated players
competitions settings. participated in the 2 matches. In both competition settings,
The effect of acute stress and the impact of sustained the players were encouraged to drink water ad libitum during
periods of stress on immune function also have been the break periods to maintain hydration levels. Food intake on
considered and focused by researchers. Jemmot et al. (15)
each day was not strictly monitored during this study, but
showed that chronic psychological stress appears to lower
players were instructed to maintain their normal dietary
salivary immunoglobulin A (SIgA) level. Deinzer et al. (6), intake on each day of testing. Session-RPE was obtained after
corroborating with these findings, demonstrated that sus- 30 minutes of each match from all the players involved
tained periods of psychological stress have been asso- in the study. Saliva samples were also collected before and
ciated with a reduction in SIgA concentration. The SIgA is after each match and during a rest day (baseline; BL) at the
the most commonly studied marker of the mucosal immunity same time of the prematches warm-up (PRE) (;18:30 hours),
(3). There is evidence to indicate that reduced levels of SIgA 1 week before the RM.
are related to increased risk of upper respiratory tract
infection (URTI), which could affect the performance in Subjects
sports (3). Thus, the association among acute and chronic Initially, 14 young elite male volleyball players volunteered
stress, SIgA, URTI, and sports performance should be to participate (mean 6 SD: age, 19 6 0.3 years; height, 196 6
addressed during real competitive sports setting. 6 cm; and body mass, 85 6 8 kg). The data from 2 athletes
Despite these efforts to compare the stress responses who did not participate in both investigated matches were
between different sports environments using some distinct excluded. Thus, data from 12 athletes were retained for
parameters, most of the studies, particularly in team sports, analyses. The athletes belonged to an under-19 team playing
have not addressed the impact of match importance (e.g., in the main Sao Paulo (Brazil) volleyball competition. On
regular match, semifinal, or final championship matches) on a weekly basis, each player typically trained twice a day
perceptual, hormonal and mucosal immune parameters. It (90120 minutes per session), 5 dwk21, and played in 1
could be speculated that greater perceptual responses and official match. The training sessions consisted of volleyball
greater hormonal and mucosal immune changes would be drills, tactics and specific conditioning work as well as weight
observed after playoff (decisive; more important) matches training and plyometrics. The participants provided informed
compared to regular matches. Monitoring these responses consent before the study commenced. All procedures
could help coaches to plan appropriate loads during the received local ethics committee approval.
preparation to decisive matches reducing the likelihood to
increments on negative affective properties and risk to URTIs, Session Ratings of Perceived Exertion
which in turn might affect the performance. Session-RPE was used as an indicator of exercise intensity.
Therefore, the purpose of this study was to compare the The session-RPE assessment was conducted as previously
session-RPE, salivary cortisol (sC) and SIgA responses with described by Foster (11). Briefly, this method of monitoring
2 official matches (regular season match vs. final champion- training and competition load required each athlete to
ship match) in elite young male volleyball players. It was provide a RPE using the CR-10 scale (5,11). These data were
hypothesized that greater responses would be observed collected 30 minutes after each match to ensure that the
in the final official volleyball match as compared to regular perceived exertion was based on the entire match rather than
season official match. the last match effort. To assess the match intensity, the

VOLUME 27 | NUMBER 1 | JANUARY 2013 | 203

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Match Outcomes in Volleyball Players

athletes were asked individually, a simple question: How plate were decanted and washed 5 times with 250 ml of the
was your workout? and a chart was shown that outlined the ELISA wash buffer to remove all unbound substances. After
full RPE scale with the appropriate explanations. The data washing, 100 ml of tetramethylbenzidine (TMB) substrate
were recorded by the same investigator on both occasions. solution was added and incubated for 5 minutes at room
The study group was familiarized with the use of the session- temperature with no mixing. This enzyme acted on the
RPE before the beginning of the experiment. substrate and caused a blue color to appear in proportion to
the amount of peroxidase present. Finally, 50 ml of the stop
Saliva Sampling and Analysis solution was added to the wells and the optical density
The subjects provided saliva samples 30 minutes before the was read on the plate reader at 450 nm. A yellow color
prematch warm-up (PRE) (;18:30 hours), and postmatch was formed after stop solution was added. The amount of
(POST) samples were collected within 10 minutes of the color detected was directly proportional to the amount of
completion of each match (;20:30 hours). Each subject sat SIgA present. From a calibration curve (optical density
with eyes opened, head tilted slightly forward, and making versus IgA concentration of the calibrators) the concentra-
minimal orofacial movements. The subjects abstained from tion of SIgA (micrograms per milliliter) in each sample was
food and caffeine products for at least 2 hours before the
collection of saliva. Unstimulated saliva was collected into
a preweighed sterile 15-ml centrifuge tube over a 5-minute
period. The tubes were reweighed before analysis, so that
saliva volume could be estimated. Saliva density was assumed
to be 1.00 gml21. Immediately after collection, the saliva
samples were frozen and stored at 280 C until assaying for
sC and SIgA concentrations. The SIgA and sC concen-
trations were measured in duplicate using enzyme-linked
immunosorbent assays (ELISA) in accordance with previous
studies (2123). Briefly, SIgA concentration was measured by
ELISA (SIgA EIA kit, ALPCO Diagnostics, Salem, MA,
USA). Saliva samples were thawed, centrifuged at 1,630g for
10 minutes, and the supernatant was diluted (1:2,000) in
ELISA wash buffer. Subsequently, 100 ml of calibrators and
diluted saliva samples were then added to microtiter wells
(precoated with polyclonal rabbit antihuman IgA) and
incubated for 1 hour, with constant shaking, at room
temperature. After incubation, the plate was aspirated and
washed 5 times with 250 ml of ELISA wash buffer to remove
all unbound substances. Then, 100 ml of peroxidase-labeled
mouse anti-IgA conjugated was added to each well on the
microtiter plate. After incubating the plate for 1 hour, with
constant shaking at room temperature, the contents of the

Figure 2. A) Salivary cortisol concentrations (mean and SD) during the

investigated conditions (regular season match [RM], final championship
match [FM], and baseline); a = significant differences to RM prevalue and
Figure 1. Session-rating of perceived exertion (RPE, AU) in regular baseline; b = significant difference to RM postvalue and baseline (p ,
season match (RM) and final championship match (FM); a = significant 0.05). B) Individual salivary cortisol (sCortisol) to pre-RM and pre-FM. C)
difference to RM (p , 0.05); AU = arbitrary units. Individual salivary cortisol (sCortisol) to post-RM and post-FM.

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Greater Session-RPE was observed for FM as compared with
RM (t = 23.416; p , 0.01). The mean values (SD) were
6.5 (1.0) and 5.9 (1.4) in FM and RM, respectively (Figure 1).
The sC responses can be observed in Figure 2 (RM, FM, and
BL). A significant increase in sC (pre and post) was observed
in the FM as compared with RM. Moreover, significant
higher values were verified in FM as compared with BL. The
SIgAabs data are presented in Figure 3. Significant differences
were detected between the conditions. The SIgAabs concen-
tration to pre-FM was significantly lower than the pre-RM
and BL. No changes were observed for SIgArate.

Figure 3. The SIgAabs concentration during the investigated conditions

(regular season match [RM], final championship match [FM], and
baseline); b = significant differences to RM prevalue and baseline. SIgA = The aim of this study was to compare the session-RPE scores
salivary immunoglobulin A.
and the salivary immune-endocrine responses between one
regular season match (RM) and one final championship match
(FM) in elite young volleyball players. It was hypothesized
interpolated. The SIgA secretion rate (micrograms per
that greater changes would be observed in the FM compared
minute) was calculated by multiplying the absolute SIgA
with RM because of the match importance. In agreement to
concentration by salivary flow rate (milliliter per minute).
the initial hypothesis, a greater session-RPE score was observed
Salivary cortisol concentrations were also measured in
for the FM. It was also detected greater cortisol concentra-
duplicate by ELISA (ALPCO Diagnostics, Salem, MA,
tions during the FM, indicating that stress-related hormone
USA) Saliva samples were thawed and centrifuged at 1,630g
responses were increased by the match importance as com-
for 20 minutes. Then, 50 ml of calibrators, control and
parison with RM. In addition, lower SIgA absolute concentra-
supernatant saliva samples were added to microtiter
tion was observed in FM in the prematch moment.
wells (precoated with rabbit anticortisol antibody) and, in
In this study, the match intensity was assessed by means of
sequence, 100 ml of cortisol-horseradish peroxidase conju-
the session-RPE, as proposed by Foster (11). This method has
gate concentrate was added to the same wells and incubated
been largely used in exercise and sports settings, and previous
for 45 minutes, with constant shaking (250 rpm), at room
investigations have shown its validity (1,5,14,18,20). The
temperature (25 C). After incubation, the plate was aspirated
greater session-RPE scores verified in FM suggest that the
and washed 3 times with 300 ml of diluted wash buffer per
match importance affect the perception of effort in volleyball
well to remove all unbound substances. Then, 150 ml of TMB
players. It is important to note that the investigated matches
substrate solution was added and incubated on a plate shaker
were performed against the same opponent. Thus, it is rea-
for 15 minutes at room temperature. Finally, 50 ml of the stop
sonable to admit that the level of the opponent team was not
solution was added to the wells, and the absorbance was read
a confounding factor comparing both matches. The physical
on the plate reader at 450 nm (ELX 800VVUniversal
demands, challenges, strengthen, and weakness faced in the
Microplate Reader, Bio-TeK instruments, USA). A yellow
RM and FM were probably similar between matches, but the
color was formed after the addition of stop solution. From
match importance was distinct.
a calibration curve (absorbance vs. cortisol concentration of
Despite the greater session-RPE verified in FM, no
the calibrators) the concentration of cortisol in each sample
significant changes in sC from premoment to postmoment
was interpolated. The test-retest correlation of the SIgA and
was observed. However, greater cortisol concentrations were
sC in our laboratory is between 0.96 and 0.98. The coefficient
noted for FM as compared with RM in both premoments and
of variation for both sC and SIgA was ,6%.
postmoments. This greater cortisol value observed before the
Statistical Analyses FM as compared with the RM and baseline conditions
Data distribution was assessed using the Shapiro-Wilk test. suggests the occurrence of the anticipatory effect phenom-
A 2-way analysis of variance (condition vs. moments) enon. Other studies have consistently reported the anticipa-
with repeated measures was used to compare the salivary tory effect in different sports. For example, Filaire et al. (10)
parameter levels. Tukeys honestly significant difference test observed higher resting cortisol concentrations during
was used as the post hoc procedure. In the case of violation of competition days compared with control days and higher
the assumption of sphericity, the significance was established values before the first interregional match compared with
by using the Greenhouse-Geisser procedure. A paired t-test those measured before the first regional competition match.
was used to compare the Session-RPE responses between Previous investigations in team sports related to the
RM and FM. The level of significance was set at p , 0.05. anticipatory increases of stress hormones (2,9) have typically

VOLUME 27 | NUMBER 1 | JANUARY 2013 | 205

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Match Outcomes in Volleyball Players

reported data comparing samples taken before the start of a investigations assessed the SIgA responses in elite young
competition with samples obtained in a noncompetition day volleyball players during official matches. The present find-
(9). However, there are no previous studies comparing this ings are in accordance with those of other studies that
phenomenon in distinct periods of the competitive season, investigated SIgA responses in team sports. For example, no
specifically concerning official matches in elite male volleyball changes in salivary IgA levels after an 80-minute collegiate
players. Thus, this study seems to be the first in team sports to rugby (16) or 70-minute soccer matches (21) were reported.
compare 2 matches in different phases of the competitive Conversely, Moreira et al. (24) demonstrated that simulated
season in male volleyball players competing against the same futsal matches induced decreases in salivary IgA levels in
opposing team and also considering a control condition (BL). professional futsal players. Some differences in sports
Notably, the investigated team was defeated in both demands and characteristic between the modalities might
occasions. The nonsignificant elevations in cortisol values explain these controversial responses. The physical demands
from precompetition to postcompetition might be explained, (intermittent nature and no physical contact) of a volleyball
at least in part, by this fact. Earlier studies showed greater match, which includes significant periods of recovery
increases in cortisol from precompetition to postcompetition between efforts and relatively short durations of action,
in winners compared with losers (8). Doan et al. (8) pointed could explain the lack of changes in mucosal parameters.
out that, despite not knowing the mechanisms involved in This explanation suggests that official volleyball matches do
this association, better performers (winners) might be more not affect the mucosal immunity, even when elevated cortisol
concerned about their performance, whereas losers may levels are observed, as was verified in the FM.
be more relaxed. However, this speculation needs to be The main limitation of this study is the lack of other
confirmed in future studies. measures of physiological parameters during both matches
In regard to the mucosal parameters, it was not observed (i.e., heart hate, lactate). Time motion analysis could also reveal
any acute changes in SIgAabs or SIgArate in RM and FM, from differences in the number and intensity of actions and distance
pre-to-post match. However, lower prevalues (SIgAabs) were covered by the players and could help to explain differences in
verified in FM as compared with RM and control condition. session-RPE recorded 30 minutes after the games. Nonethe-
These findings suggest that psychosocial factors related to less, these variables cannot be controlled for during a real
the preparation for a decisive match might have affected the world setting as employed in this study. Fortunately, the
mucosal immune function. number of sets played was similar between conditions.
Several authors have been demonstrated such a relation- In summary, the results of this study showed greater RPE
ship, for instance, Jemmott et al. (15) reported that chronic and cortisol levels in the FM when compared with the RM,
psychological stress induced lower SIgA level. Indeed, suggesting that perceptual and hormonal response might
sustained periods of psychological stress have been associ- be influenced by the match importance. In addition, the
ated with a reduction in SIgA concentration (6). Moreover, anticipatory effect observed in the FM confirms that the
Moreira et al. (22) showed that a 17-day preparation for an greater level of involvement affects hormonal responses in
international basketball championship reduces the SIgA a sports setting. Moreover, the preparation to a decisive
levels for both athletes and staff members, indicating that not match might influence the mucosal immune function,
only the training load, but also the psychological stress could possibly because of increments in psychological stress.
be responsible for the changes in the mucosal parameters.
It is unlikely that the greater prematch cortisol levels PRACTICAL APPLICATIONS
verified in the FM induced the decrement of SIgA. Possibly, According to the present outcomes, on season training
the modifications of IgA synthesis might explain the lower monitoring of perceptual, hormonal and mucosal immune
SIgA values observed in pre-FM (3). Moreover, it is also responses can provide valuable information regarding the
reasonable to assume that the preparation (training days magnitude of stress induced by sports training and com-
before the match) for the final match (short-term chronic petitions and how athletes cope with sports induced stress.
stress) would have induced the lowered SIgA values, rather Coaches and athletes, if possible, may use the sC and SIgA
than the possible effect of the anxiety and negative affective responses as an approach to monitor changes in affective state
state, that could have been experienced by the athletes just and its effect on mucosal immunity during distinct micro-
before the commencement of the match. These assumptions cycles within the training process. Moreover, monitoring
can be supported by the findings from some authors who internal loads by means of the session-RPE method might be
have presented evidence that the acute psychological stress useful to identify the demands from official competitive
leads to increments and not to decrements in SIgA as matches. The precise monitoring of these responses could
observed in this study (28,29). help coaches to avoid excessive training loads during the
Regarding the nonsignificant acute responses (pre-to-post preparation to decisive matches. The proper periodization of
match) in SIgA in the present investigation, it is important to training loads may reduce the likelihood to decrements on
note that few studies have been conducted during actual mucosal immunity and its consequently risk to URTIs, which
competition with team sport athletes. In fact, no previous in turn might affect the performance.
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ACKNOWLEDGMENTS 15. Jemmott, JB, Borysenko, JZ, Borysenko, M, McClelland, DC,

Chapman, R, Meyer, D, and Benson, H. Academic stress, power
The authors would like to thank the Fundacxao de Amparo a` motivation, and decrease in secretion rate of salivary secretory
Pesquisa do Estado de Sao Paulo (Sao Paulo Research immunoglobulin A. Lancet 321: 14001402, 1983.
Foundation, process, 2008/10404-3) for funding this research. 16. Koch, AJ, Wherry, AD, Petersen, MC, Johnson, JC, Stuart, MK, and
Sexton, WL. Salivary immunoglobulin A response to a collegiate
They also wish to acknowledge all volleyball players, their
rugby game. J Strength Cond Res 21: 8690, 2007.
coach, Nivaldo Starnini Adegas and research support staff
17. Lazarus, RS and Folkman, S. Stress, Appraisal, and Coping. New York:
involved in this study for their committed participation. Springer Publishing Company, 1984.
18. Manzi, V, DOttavio, S, Impellizzeri, FM, Chaouachi, A,
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