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Research Centre in Physical Activity, Health and Leisure, University of Porto, Portugal
Department of Sport Biology, Faculty of Sport Sciences, University of Porto, Portugal
c
Department of Soccer, Faculty of Sport Sciences, University of Porto, Portugal
d
Department of Clinical Analysis, Faculty of Pharmacy, University of Porto, Portugal
e
Institute for Molecular and Cell Biology, University of Porto, Portugal
Received 11 December 2007; received in revised form 3 April 2008; accepted 8 April 2008
Available online 23 April 2008
Abstract
Background: Exercise is a prone condition to enhanced oxidative stress and damage and the specific activity pattern of a soccer match may
favour additional pro-oxidant redox alterations. To date, no studies have reported the impact of a soccer match on oxidative stress and muscle
damage markers.
Aim: To analyse the effect of a competitive soccer match on plasma levels of oxidative stress and muscle damage markers, and to relate these
findings with lower limb functional data.
Methods: Blood samples, leg muscle strength, sprint ability and delayed-onset muscle soreness (DOMS) were obtained in 16 soccer
players before, at 30 min, 24, 48 and 72 h after a soccer match. Plasma creatine kinase (CK), myoglobin (Mb), malondialdehyde (MDA),
sulfhydryl (SH) groups, total antioxidant status (TAS), uric acid (UA) and blood leukocyte counts were determined.
Results: A soccer match elevated plasma Mb following 30 min and CK levels throughout the 72 h-recovery period. MDA increased
throughout the recovery period and SH decreased until 48 h post-match. TAS increased at 30 min and UA increased throughout the 72 h
recovery. Blood neutrophils increased at 30 min whereas lymphocytes decreased and returned to baseline from 24 to 72 h. DOMS was higher than
baseline until 72 h. Lower limb strength and sprint ability were lower than baseline until 72 h recovery.
Conclusion: The present data suggest that a soccer match increases the levels of oxidative stress and muscle damage throughout the 72 hrecovery period. The extent to which the redox alterations are associated with the recovery of muscle function should be further analysed.
2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: Football game; Antioxidants; Strength; Sprint
Introduction
Reactive oxygen and nitrogen species (RONS) have the
potential to react with a variety of chemical compounds, being
0009-9120/$ - see front matter 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2008.04.008
842
soreness and contraction-induced muscle injury has been recently reported [for refs see [2]].
The activity of soccer players during the competitive season
entails 1 week cycles of training, taper, competition and recovery.
At the top level, this cycle is altered by several irregularities in the
competitive fixture list, being match day not necessarily the same
from 1 week to another. Moreover, players from top level teams
may be involved in additional commitments such as national
cups and other knock-out matches, or representing their countries in international championships. These competitive demands
may impose strains to various physiological systems, including
musculoskeletal, nervous, immune and metabolic, to a point
where recovery strategies post-exercise became influential in
preparing for the next match [3].
The high absolute levels of mitochondrial oxygen consumption, the increased circulating catecholamine, the elevated participation of eccentric muscle contraction-induced damage
and inflammatory response, the intermittent and repeated sprint
actionscausing temporary ischemiareperfusion events in
skeletal muscle are plausible factors that may influence RONS
production during and after a soccer effort. Thus, and despite
chronic exercise training having a protective effect through
improvement of antioxidant capacity [4,5], it is likely that
training sessions as well as the competitive matches expose
participants to oxidative stress and damage with consequent
muscle damage, both during, immediately post-exercise and
throughout the recovery.
Although scarce data have been published regarding the
effects of oxidative stress on exercise performance, there is a
possibility that prior oxidative damage caused by intensive
training periods and/or oxidative modifications while exercising
might compromise the healthy status of the players as well as
exercise performance [6].
Several reports focused on some stress biomarkers, including those of oxidative damage, as well as on the antioxidant status of soccer players under regular training periods
that have been provided [4,5,7]. However, despite postexercise vitamin C supplementation failing to attenuate leg
muscle dysfunction, plasma malondialdehyde, interleukin-6,
creatine kinase and myoglobin increase during the 72 h-recovery period after 90 min of a shuttle running designed to
correspond to the average exercise intensity of playing soccer
[8], no data have been provided regarding the impact of a
soccer match on oxidative stress and damage markers so far.
Moreover, the impact of a soccer match on muscle damage
markers throughout the post-game recovery period has also
been scarcely studied.
The understanding of the redox-based alterations imposed by
a soccer match can contribute with additional physiological
knowledge on the effects of a soccer game on players and
particularly, to improve possible recovery strategies based on
possible antioxidant supplementation. In this regard, the purpose
of the present study was to determine the impact of a soccer
match on the recovery of plasma markers of oxidative stress and
muscle damage in the 72 h-post-match. Leg muscle functional
data, sprint ability, muscle damage as well as leukocyte counts
were also determined throughout the same period.
Methods
Subjects
Sixteen male soccer players from secondary divisions
participated in this study after being informed about the aims,
experimental protocol, procedures and after delivering written
consents. The experimental protocol was approved by the
Ethical Committee of the Faculty of Sport Sciences, University
of Porto, Portugal, and followed the Declaration of Helsinki of
the World Medical Association for research with humans.
Experimental design and procedures
For 2 weeks prior to data collection and during the protocol
period, soccer players were instructed not to change their
normal eating habits and to refrain from additional vitamin or
antioxidant dietary supplementation. Subjects were also
instructed to abstain from exhaustive exercise during the 72-h
pre- and post-match, with exception of functional tests. Subjects
visited the lab 5 days prior the match to determine maximal
oxygen uptake and maximal heart rate.
Blood samples and functional data (quadriceps and hamstrings muscle strength and 20 m sprint ability) were assessed
pre-match and 30 min, 24, 48 and 72 h of the recovery period in
response to a competitive (2 45 min) soccer match. On the day
of the game, players arrived at the laboratory after an overnight
fast of between 10 and 12 h. A resting blood sample was taken
after subjects had been standing for at least 15 min, after which
subjects consumed a light standardized meal and drink and
rested for 2 h. The meal consisted of 1.7 g white bread and 0.3 g
of low-fat spread; both values are per kilogram of body mass
[8]. Pre-match muscle strength and sprint ability were assessed
during the 2 h period between the consumption of the preexercise meal and the start of the soccer match.
For 3 days after the match, subjects returned to the laboratory
after an overnight fast and at approximately the same time of the
morning (within 1 h). A blood sample was taken from the
forearm vein after the subjects had been at complete rest for at
least 15 min. Subsequently, the players performed the strength
and speed tests as outlined below.
Maximal oxygen uptake and heart rate
Five days prior the match, the subjects performed an incremental treadmill (Quasar-Med, Nussdorf, Germany) test
until voluntary exhaustion to determine maximal oxygen uptake
(VO2max) and maximal heart rate (HRmax). Expired respiratory gas fractions were measured using an open circuit breathby-breath automated gas-analysis system (Cortex, Metalyzer,
3 B, Leipzig, Germany). HR was measured using a HR monitor
(Vantage NV, Polar Electro, Kempele, Finland).
Intensity of the match
Heart rate was measured during the match and recorded
every 5 s using a HR monitor (POLAR TEAM SYSTEMTM, Polar
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20 m sprint ability
Sprint ability measurements were carried out using telemetric
photoelectric cells placed at 0 and 20 m (Brower Timing System,
IRD-T175, Utah, USA). The players stood 1 m behind the
starting line, started on a verbal signal being time activated when
players cross the first pair of photocells, and then ran as fast as
they could to complete the 20 m distance. Players completed two
runs and the best time at each distance was registered.
Blood sampling and preparations
All the venous blood samples were taken by conventional
clinical procedures using EDTA as anticoagulant. Nevertheless,
no tourniquet constriction was used in order to minimize potentially enhanced oxidative stress induced by an ischemia
reperfusion maneuver.
An aliquot of the whole blood was used to perform leukocyte
counts. The remaining freshly withdrawn blood was immediately centrifuged at 3000 rpm during 10 min for careful removal
of the plasma. Plasma was separated into several aliquots
and rapidly frozen at 80 C for later biochemical analysis of
myoglobin (Mb), creatine kinase (CK), total antioxidant status
(TAS), uric acid (UA), malondialdehyde (MDA) and protein
sulfhydryl groups (SH).
Biochemical assays
Strength assessment
In order to evaluate muscle function, maximal gravity corrected concentric peak torque of quadriceps and hamstrings were
measured during isokinetic knee joint movement of the
dominant leg at an angular velocity of 90.s 1 (1.57 rad.s 1)
according with [11] using an isokinetic dynamometer (Biodex
System 2, NY, USA). After individual self-report, the dominant
leg was determined by a routine visual inspection in a simple
target kicking test requiring accuracy according to the procedures described elsewhere [12].
Briefly, the subjects were familiarized with the muscle function test on at least two occasions during preliminary visits to the
laboratory. They were seated on the dynamometer chair at 85
inclination (external angle from the horizontal) with stabilization
straps at the trunk, abdomen and thigh to prevent inaccurate
joint movements. The knee to be tested was positioned at 90 of
flexion (0 = fully extended knee) and the axis of the dynamometer lever arm was aligned with the distal point of the lateral
femoral condyle. Before the anatomical alignments and
procedures, all the subjects were instructed to kick and also to
bend the tested leg as hard and fast as they could through a
complete range of motion (from 90 to 0). Subjects were also
instructed to hold their arms comfortably across their chest to
further isolate knee joint flexion and extension movements.
Prior to muscle function measurements, subjects were taken to
a standardized warm-up consisting of 5 min of gentle running and
stretching. All subjects performed a specific sub-maximal warmup protocol on the isokinetic device. Three maximal repetitions at
angular velocity 90.s 1 (1.57 rad.s 1) were therefore carried out.
Plasma creatine kinase (CK) activity was determined spectrophotometrically using a commercial test kit (ABX A11A01632,
Mompelier, FR). Plasma myoglobin concentration was assessed
using a commercial test kit (myoglobin bioMerieux 30446,
Carnaxide, PT).
Plasma MDA was assayed according to the method described
by Rohn et al. [13] with some modifications and measured by the
formation of thiobarbituric acid reactive substances at 535 nm.
Briefly, plasma was incubated, at 25 C, in 500 L of a medium
consisting of 175 mM KCl, 10 mM Tris, pH 7.4. Samples of
50 L were taken and mixed with 450 L of a TBARS reagent
(1% thiobarbituric acid, 0.6 N HCl, 0.0056% butylated
hydroxytoluene). The mixture was heated at 8090 C during
15 min, and re-cooled in ice for 10 min before centrifugation in
Eppendorf centrifuge (1500 g, 5 min). The amount of TBARS
formed was calculated using a molar extinction coefficient of
1.56 105 M 1 cm 1 and expressed as nanomoles of MDA per
milligram of protein [14].
Oxidative modification of protein SH groups in plasma
was quantified by spectrophotometric measurement at 414 nm
according to the method proposed by Hu [15]. Briefly, the
colorimetric assay was performed after the reaction of 50 L
aliquot of plasma with 10 L of 5,5-dithio-bis (2-nitrobenzoic
acid) (10 mM) in a medium containing 150 L of Tris (0.25 M)
and 790 L methanol, at 414 nm against a blank test. SH
content was expressed in nmol/mg of plasma protein (414 =
13.6 mM 1 cm 1 ).
TAS was measured spectrophotometrically using a commercial kit (Randox NX2332 Crumlin, UK). The assay is based on
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Table 1
Anthropometric and physiological characteristics of the subjects
Variables
Mean SD
Age (yr)
Mass (kg)
Height (cm)
% Body fat
VO2max (mL.Kg 1.min 1)
Hrmax (bpm)
21.3 1.1
70.7 6.3
175.0 6.0
8.3 1.9
55.1 5.1
196.0 7.0
Table 2
Frequency, mean duration and percent of match time spent on the considered motor categories
Frequency (n)
Mean duration (min)
% total time
Values are mean SD.
Standing
Walking
Jogging
Cruising
Sprinting
Backwards running
Sideways running
114 44.8
7.0 2.5
7.8 3.4
408.6 93.8
39.5 3.6
43.8 7.9
441.9 96.2
31.8 7.4
35.3 5.6
69.6 10.4
2.9 1.5
5.8 2.3
41.7 18.0
2.2 1.5
2.5 1.3
122.1 26.6
4.3 1.3
4.8 1.9
64.9 4.8
1.4 0.4
1.6 0.6
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Fig. 1. Sprint ability evaluated as running time under 20 m before and throughout the 72 h-post-match recovery period. Values are means SEM. vs. Pre (p b 0.05).
Fig. 2. Dominant leg quadriceps (2A) and hamstrings (2B) peak torques evaluated before and throughout the 72 h-post-match recovery period. Values are means SEM.
vs. Pre (p b 0.05).
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Fig. 3. Delayed onset muscle soreness before and 30 min, 24, 48 and 72 h after a soccer match. Values are means SEM, vs. Pre (p b 0.05).
both at low and high intensity activities were also similar to that
described for players of the same level, and below to those
observed in elite players [9]. These observations may suggest
that the analysed match intensity was somewhat similar to other
non-elite games, and probably lower than the intensity
performed by elite soccer players.
As an estimated 15% of the total VO2 results in the forU
mation of O2 [18] and given the high level of VO2 accompanying soccer, it is not surprising that the biomarkers of
oxidative stress and damage had increased. In addition, other
concurrent factors can influence cellular and blood antioxidant
status. For example, stress hormones undergoing autoxidation
[19] and circulating neutrophil-induced oxidative burst [20,21]
can contribute to the observed blood oxidative stress and
damage. The influence of eccentric exercise-mediating muscular
damage-like events on the formation of RONS has also been
reported [22]. Considering the specific physiological demands
imposed by a soccer match, none of these potential RONS
sources should be ruled out in the current study. However, it is
important to note that under the technical constrains of the
present study we cannot conclusively demonstrate a casual link
between any of those potential sources and the increased plasma
oxidative stress and damage found.
The increased oxidative damage induced by the soccer
match can be observed by the additional accumulation of lipid
peroxidation by-products, measured as plasma MDA (Fig. 7).
Accordingly, the game also induced a significant decrease in
plasma sulphydryl residues (Fig. 7), indicating increased
disulphide linkages (SS) from both proteins and reduced
glutathione (GSH). It is important to highlight that the increase
in the levels of oxidative damage after the match occurred
despite the possible up-regulation of antioxidant systems
previously observed under rest conditions in players regularly
involved in training and competition when compared with
sedentary controls [4,5]. Moreover, as soccer players undergoing
regular training showed higher plasma oxidative stress and
damage levels than sedentary controls [4,5] and considering
the present data assessed in regularly trained subjects, it would
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Fig. 4. Plasma Mb (4A) and CK (4B) levels before and 30 min, 24, 48 and 72 h after a soccer match. Values are means SEM, vs. Pre (p b 0.05).
848
Fig. 5. Plasma TAS (A) and UA (B) levels before and 30min, 24, 48 and 72h after soccer match. Values are means SEM, vs. Pre (b 0.05).
observed that, even in a study in which high levels of apoptosisinducing factors were generated, such as cortisol and isoprostanes, lymphocyte apoptosis did not contribute to post-exercise
lymphocytopenia, others suggested that apoptosis may partially
account for the transient loss of lymphocytes after intense exercise with consequent immunosuppression [32]. Moreover,
hormonal changes such as catecholamine over production during exercise have been described to be responsible for inducing
apoptosis [34].
As indirect evidence of delayed-onset muscle damage
induced by the soccer match we considered the levels of muscle
soreness (Fig. 3), the lower limb muscle strength (Fig. 2), the
appearance of the muscle proteins creatine kinase and
myoglobin (Fig. 4) in plasma and the counts of blood inflammatory cells over a 72 h period after the match. In the
present study, the magnitude of changes induced by the soccer
match in these parameters was rather lower than those observed following other specific models of exercise-induced
muscle damage, such as repeated maximal eccentric contractions, which were reported to be severely affected [22]. Expectedly, the significant alterations observed after the soccer
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Fig. 6. Changes in blood leukotype, neutrophil and lymphocyte counts before and throughout the 72 h-post match recovery period. Values are means SEM, vs.
Pre (b 0.05).
Fig. 7. Plasma MDA (A) and -SH (B) levels before and 30min, 24, 48 and 72h after soccer match. Values are means SEM, vs. Pre (b 0.05).
850
match were somewhat close to some reported after an intermittent exercise protocol designed to simulate a soccer match
play [8,3537].
Several reports [38,39] about the mechanisms related to
delayed-onset muscle damage have demonstrated that RONS
not only directly causes damage by oxidation of macromolecular
cellular components such as lipids, proteins and DNA, but also
acts as a regulator of inflammation. Increased RONS production
promotes the translocation to the nucleus of certain redox-sensitive transcription factors, regulating inflammatory mediators
such as cytokines, chemokines and adhesion molecules. Therefore, increased RONS production due to exercise is possibly
involved in delayed-onset muscle damage associated with
phagocyte infiltration secondary to the increased expression of
inflammatory mediators. However, given that the soccer match
did not represent a sufficient severe muscular stimulus to cause
leukocyte infiltration, as shown by the maintenance of blood
leukocyte counts from 24 to 72 h-recovery period when compared to baseline (Fig. 6), the possible effects of neutrophilrelated oxidative burst on muscle damage induced by soccer
should probably be ruled out. This lack of variation in blood
leukocyte counts, with the significant increase in the plasma
levels of oxidative damage that had occurred in the present study
was initially not expected. It is likely that the contribution of
other RONS sources during the post-exercise periods might be
considered, such as monocyte and macrophage oxidative burst
[40]. It is possible that a delayed and continuous monocyte
mobilization from bone marrow, thus compensating possible
infiltration of these cells into muscle tissue after damaging
exercise [40,41] had occurred in the present study masking
leukocyte count changes in blood. This delayed inflammatory
response may be responsible for amplifying and/or repairing
skeletal muscle injury [41].
Actually, other adaptive responses induced by contractile
activity in general, and by soccer muscular effort in this particular case, appear to be directly mediated by RONS as signaling molecules in the activity and/or expression of several
transcription factors such as HSF1, NFkB and AP-1 [42,43],
which are potentially involved in the up-regulation of cellular
defense against deleterious stress conditions, including muscle
damage. Animal studies demonstrated that the overexpression of
skeletal muscle heat shock proteins (HSPs) is associated with
an increased superoxide production during non-damaging contraction and a subsequent transient and reversible oxidation of
protein thiol groups within the muscle [44], a signal for the
activation of HSP-mediated adaptive response. These data were
supported by evidence that the increased HSP content that
occurs following an acute period of non-damaging exercise in
humans was attenuated by prior vitamin C supplementation [45].
Moreover, inhibition of free radical production after damaging
exercise (downhill running) by ascorbic acid supplementation
did not affect muscle soreness and delayed the recovery of
muscle function [46]. Extrapolating this hypothesis to a situation
where exercise-induced muscle damage occurred such as a
soccer match, cellular pro-oxidant redox changes, particularly in
mild levels, might hypothetically have little direct effect on
the scale of muscle dysfunction, but may stimulate signaling-
mediated adaptive response through the recovery period following exercise [2].
Acknowledgments
We would like to thank the soccer players involved in
the study for their committed participation. The excellent technical and practical assistance and skilful involvement of
Sergio Ribeiro, Joo Renato, Ricardo Ladeira, Brbara Duarte,
Henrique Reguengo, and camera operators is appreciated.
The authors are grateful to the City Council of Maia for
providing the pitch where a soccer match was carried out.
Antnio Ascenso is supported by a grant from the Portuguese
Foundation for Science and Technology (SFRH/BPD/42525/
2007).
References
[1] Halliwell B, Gutteridge JM. Free radicals in biology and medicine. Oxford:
Clarendon Press; 1999.
[2] Close GL, Ashton T, McArdle A, Maclaren DP. The emerging role of free
radicals in delayed onset muscle soreness and contraction-induced muscle
injury. Comp Biochem Physiol A Mol Integr Physiol 2005;3:25766.
[3] Reilly T, Ekblom B. The use of recovery methods post-exercise. J Sports
Sci 2005;6:61927.
[4] Cazzola R, Russo-Volpe S, Cervato G, Cestaro B. Biochemical assessments
of oxidative stress, erythrocyte membrane fluidity and antioxidant status
in professional soccer players and sedentary controls. Eur J Clin Invest
2003;10:92430.
[5] Brites FD, Evelson PA, Christiansen MG, Nicol MF, Basilico MJ, Wikinski
RW, et al. Soccer players under regular training show oxidative stress but an
improved plasma antioxidant status. Clin Sci (Lond) 1999;4:3815.
[6] Vollaard NB, Shearman JP, Cooper CE. Exercise-induced oxidative
stress: myths, realities and physiological relevance. Sports Med 2005;12:
104562.
[7] Banfi G, Malavazos A, Iorio E, Dolci A, Doneda L, Verna R, et al. Plasma
oxidative stress biomarkers, nitric oxide and heat shock protein 70 in
trained elite soccer players. Eur J Appl Physiol 2005;5:4836.
[8] Thompson D, Williams C, Garcia-Roves P, McGregor SJ, McArdle F,
Jackson MJ. Post-exercise vitamin C supplementation and recovery from
demanding exercise. Eur J Appl Physiol 2003;34:393400.
[9] Mohr M, Krustrup P, Bangsbo J. Match performance of high-standard
soccer players with special reference to development of fatigue. J Sports
Sci 2003;7:51928.
[10] Duarte JA, Magalhaes JF, Monteiro L, Almeida-Dias A, Soares JM, Appell
HJ. Exercise-induced signs of muscle overuse in children. Int J Sports Med
1999;2:1038.
[11] Magalhaes J, Oliveira J, Ascensao A, Soares J. Concentric quadriceps and
hamstrings isokinetic strength in volleyball and soccer players. J Sports
Med Phys Fitness 2004;2:11925.
[12] Porac C, Coren S. Lateral preferences and human behaviour. New York:
Springer-Verlag; 1981.
[13] Rohn TT, Hinds TR, Vincenzi FF. Ion transport ATPases as targets for free
radical damage. Protection by an aminosteroid of the Ca2+ pump ATPase
and Na+/K+ pump ATPase of human red blood cell membranes. Biochem
Pharmacol 1993;3:52534.
[14] Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol
1978:30210.
[15] Hu ML. Measurement of protein thiol groups and GSH in plasma. In: Parker
L, editor. Methods in enzimology. San Diego: Academic Press; 1990.
[16] Lowry OH, Rosenbrough N, Farr AL, Radall RJ. Protein measurement
with the folin phenol reagent. J Biol Chem 1951;193:26575.
[17] Krustrup P, Mohr M, Steensberg A, Bencke J, Kjaer M, Bangsbo J. Muscle
and blood metabolites during a soccer game: implications for sprint
performance. Med Sci Sports Exerc 2006;6:116574.
851