Post-Match Changes in Muscle Damage Markers among U-21 Soccer Players

Th is study aimed to investigate the eff ect of offi cial soccer matches on selected markers of muscle damage in U-21 soccer players. A group of 19 trained, healthy male soccer players from the junior category took part in this study. Blood samples were assessed pre-match and immediately aft er a match in response to a competitive (2×45 min) soccer match. Analysis was performed for muscle damage and infl ammatory markers. Signifi cant diff erences between two measures (before and aft er soccer match) exist in Aspartate-aminotransferase (AST), Lactate dehydrogenase (LDH), and Myoglobin. Plasma K+ signifi cantly decreased aft er the match (p<0.05), whereas plasma Na+ decreased slightly. Th is study showed that most selected markers of muscle damage were infl uenced by a soccer match. However, results remain inconsistent because of the infl uence of the type, duration, and intensity of exercise. Moreover, some markers show great variability among individuals.


Introduction
Regular physical activity can have signifi cant health benefi ts.However, some activities might not be healthy, due to dehydration, substrate depletion, muscle damage, infl ammation, and the increased production of free radicals.Free radicals, which are particularly produced as a result of physical exercise (Cooper, Vollaard, Choueiri, & Wilson, 2002), are involved in the process of muscle fatigue, many diseases, and aging (Finaud, Lac, & Filaire, 2006).Although there is no defi nitive conclusion on fatigue, it is assumed that two main origins are involved in this process.Th e fi rst is the central fatigue hypothesis, in which the central nervous system blocks continued extraordinary eff ort, perhaps as protection from injury (Taylor, Allen, Butler, & Gandevia, 2000).Th e second is the peripheral fatigue, in which the muscle's homeostasis has been perturbed, either through tissue damage, or some other way, to the point that the muscle is incapable of responding as eff ectively as it does when rested (MacIntosh & Rassier, 2002).Nybo et al. (2013) suggested that muscle pain, reduced power, increased creatine kinase, Myoglobin, aspartate aminotransferase and lactate dehydogenase levels are the most signifi cant indicators of muscle damage.Two studies have used very high intensity or volume of exercise to provoke muscle damage (Byrne & Eston, 2002;Davies, Rowlands, & Eston, 2009).However, match activity in soccer is infl uenced by several factors.Th e style of play can infl uence the physiological demands on players.Th e national teams of Ireland and Norway use the "direct method of play", maintaining the game at a high pace (Reilly, 1997).Th us, the fi ndings of a recent study (Gregson, Drust, Atkinson, & Salvo, 2010) indicate that match-to-match variability in performance characteristics of elite soccer players is high.Moreover, many factors within the body can infl uence muscle damage, such as infl ammation (Stoner et al., 2013), nutrition (Muñoz & Costa, 2013), intake of supplements (Goldfarb, 1999;Goldfarb, Garten, Cho, Chee, & Chambers, 2011).A signifi cant increase in muscle damage markers (Andersson, Ekblom, & Krustrup, 2008;Ascensão et al., 2008;Ispirlidis et al., 2008;Magalhães et al., 2010) has been described immediately aft er "friendly" soccer matches and throughout the post-match recovery.Ascensão et al. (2008) and more recently Fatouros et al. (2010) found noticeable muscle damage up to 72 hours post-exercise, following a soccer match.Th ere have been numerous studies on muscle damage aft er competitive matches in multiple sprint sports involving body contact, such as soccer, rugby, and fi eld hockey.However, only a few studies have evaluated muscle damage markers immediately aft er the offi cial soccer match.Th erefore, this paper aimed to estimate the markers of muscle damage in U-21 players, before and immediately aft er an offi cial soccer match.

Participants
A group of 19 trained, healthy male soccer players from the junior category of a Croatian soccer association took part in this study.Th e players were informed about the experimental procedures and possible discomforts associated with the study, and written informed consent was obtained.Th e study was approved by the Ethics Committee of the Faculty of Kinesiology, University of Zagreb and according to the Helsinki Declaration.Th e participants were aware that they could withdraw from the study at any time.Th e participant's characteristics were in average: age 20.26 ± 0.65 years, mean ± SD: body mass: 71.3 ± 5.9; body height: 1.77 ± 0.07 and maximal oxygen uptake (VO2max) 64.95±3.99ml•kg-1•min-1.Selection criteria included: (1) participation at professional (top three division leagues) level of soccer competition for at least 5 years, (2) all players participated in at least 75% of the training sessions per week and played at least 16 matches during season, (3) no consumption of exogenous anabolic-androgenic steroids or other drugs that might have aff ected their physical performance or hormonal balance during the study (for at least 6 months) (4) no recent history of febrile illness, muscle lesions, lower limb trauma, or metabolic diseases.Soccer players were instructed not to change their normal eating habits during the entire period of data collection.Nutritional supplements were not included in their diets.In addition, players were instructed to refrain from drinking beverages containing caff eine or alcohol and from consuming food in the 3 h before testing.

Blood collection and analysis
Blood samples were assessed pre-match and immediately aft er the match in response to a competitive (2×45 min) soccer match.On the day of the game, players arrived at the laboratory aft er an overnight fast of between 10 and 12 h.A resting blood sample was taken aft er participants had been standing for at least 15 min, aft er which participants consumed a light standardized meal and drink and rested for 2 h.Th e meal consisted of 1.7 g white bread and 0.3 g of low-fat spread (both values are per kilogram of body mass) (Th ompson et al., 2003).Participants abstained from alcohol and caff eine consumption for at least 24 h and did not perform any exercise for the 72 h before testing.
Fasting venous blood samples were withdrawn into heparinized tubes from a cubital vein just before and immediately aft er the soccer match, then centrifuged at 3000 rpm for 10 min to separate the plasma.Th e plasma samples were stored at -20°C until the analysis of muscle damage and infl ammatory markers.
Concerning the muscle damage and infl ammatory markers, analysis of Myoglobin, Creatine kinase (CK), Lactate dehydrogenase (LDH), Aspartate-aminotransferase (AST), C-reactive protein (CRP) were automatically performed using a Dimension Xpand Plus Analyzer (Siemens, Munich, Germany).Plasma concentrations of K+ and Na+ were determined using commercially available assays on a Dimension Xpand Plus Analyzer (Siemens, Munich, Germany).

Statistical analysis
All statistical analyses were performed using the STATISTICA v.8.0 soft ware (StatSoft Inc., Tulsa, OK, USA).Descriptive statistics and Kolmogorov-Smirnov (normality of the distribution) tests were calculated for all experimental data before inferential testing.Data were expressed as mean values ± standard deviation.A sample t-test was used to analyse where the signifi cant variations occurred.Eff ect size (ES) was classifi ed as follows: <0.2 was defi ned as trivial, 0.2-0.6 was defi ned as small, 0.6-1.2 was defi ned as moderate, 1.2-2.0 was defi ned as large, >2.0 was defi ned as very large and >4.0 was defi ned as extremely large (Hopkins, Marshall, Batterham, & Hanin, 2009).Statistical signifi cance was set at p < 0.05.

Results
As can be seen in Table 1, statistically signifi cant diff erences between two measures (before and aft er soccer match) exist in AST (p<0.05).Furthermore, LDH values were signifi cantly higher immediately aft er the match (p<0.05).No signifi cant changes were observed in plasma CK activity aft er the match (p >0.05).Th is was also the case with CRP levels, for which similar results were obtained following the match (p=0.87).Plasma Myoglobin concentrations were signifi cantly higher aft er the match in comparison to pre-match values (p < 0.05).
Table 2 shows plasma concentrations of K + and Na + measured before and aft er the matches.Plasma K + was signifi cantly decreased aft er the match (p<0.05),whereas plasma Na + gradually decreased.

Discussion
Th e purpose of the present study was to investigate the accuracy of selected markers to refl ect changes in fatigue in male U-21 players aft er an offi cial soccer match.Th e results of this study showed that signifi cant changes occurred immediately aft er an offi cial soccer match.We found that plasma LDH, AST and Myoglobin levels increased.In addition, K + levels were signifi cantly diff erent before and aft er the match.
Previous investigations have reported that a single soccer match educes signifi cant changes in muscle damage markers for as long as 48 h to 72 h post-exercise (Ascensão et al., 2008;Ispirlidis et al., 2008;Silva et al., 2013).
Published data are inconsistent for many markers of muscle damage depending on the type, duration, and intensity of exercise.For instance, Takahashi et al. (2007) reported that only LDH increased aft er a rugby match lasting 10 min, while CK, AST, and ALT remained essentially unchanged.In contrast to this, Burger-Mendonca, Bielavsky, and Barbosa (2008) showed that maximal physical exercise leads to signifi cant elevation of muscle damage markers and enzymes.Lazarim et al. (2009) analysed the changes in CK levels in 128 professional soccer players at diff erent times during the Brazilian national championship.Th e aforementioned authors identifi ed the upper limit reference value of 950IU/L as the decision limit to detect muscle overload.Recently, Hammouda, Chaouachi, Ferchichi, Kallel, and Souissi (2012) showed increased CK and LDH levels aft er the level-1 Yo-Yo intermittent recovery test in young soccer players.Accordingly, Brancaccio, Lippi, and Maff ulli (2010) consider these markers as essentially indicative of muscle damage.In this study, no signifi cant change was observed in CK levels following an offi cial soccer match in junior players.It is possible that due to the high frequency of matches and inadequate recovery soccer players can have consistently elevated CK, which was confi rmed in one study (Heisterberg et al., 2013 ).Our data show much higher values compared to the upper reference value (270 UI/L), for the general population (Mougios, 2007).Furthermore, the high variability of the measure of CK activity must also be taken into account (Halson, 2014), which was the case in this study.Research conducted on elite soccer players has also reported large variability in CK levels (Meister, Faude, Ammann, Schnittker, & Meyer, 2013).Moreover, some athletes show only small increases in CK activity due to a lower permeability of muscle cell membranes (Urhausen & Kindermann, 2002).Bearing in mind that activities of CK and Myoglobin correlate with a neutrophil response induced by stress (Suzuki et al., 1999), Myoglobin represents a useful marker to monitor the eff ectiveness of workload on muscle tissue in training (Speranza et al., 2007).Aft er a soccer match, Myoglobin may increase within 30 min (Ascensão et al., 2008), and remain increased for 72 hours, which is probably the result of low-grade infl ammation (Neubauer, König, & Wagner, 2008).Th e present fi ndings indicate that Myoglobin levels increased signifi cantly aft er an offi cial soccer match in junior professional players, which is in line with other studies.
CRP is a well-characterized biomarker of chronic infl ammation.However, it is signifi cantly infl uenced by BMI, gender, age, and smoking status (Shanely et al., 2013).Accordingly, a very small decrease in C-reactive protein levels was observed aft er the match among junior soccer players.Similarly, no relevant changes in CRP were determined following the HIIT programme (Wiewelhove et al., 2015).It appears that CRP may not be a useful muscle damage marker for monitoring fatigue and recovery immediately following a soccer match in U-21 players.However, in their study, Kostrzewa-Nowak et al. (2015) stated that CRP levels could be a valuable tool to assess the metabolic response to aerobic exercise.Because the post-match infl ammatory response of CRP levels has a 24-hour peak (Ispirlidis et al., 2008), this time-course could be considered as a valuable tool for assessing fatigue in soccer players.However, other explanations of the fatigue that occurs aft er the intense exercise periods in soccer should be sought.
During matches, some players will lose considerable quantities of electrolytes and may need to replace these during the match (Shirreff s, Sawka, & Stone, 2006).Moreover, it has been suggested that the development of fatigue during high-intensity exercise is related to an accumulation of potassium in the muscle interstitium (Bangsbo, Madsen, Kiens, & Richter;Sejersted & Sjøgaard, 2000).Th is statement was supported by the observation of muscle interstitial potassium concentrations higher than 11 mmol/l during exhaustive exercise Note: K-potassium; Na-sodium: *p < 0.05 from the corresponding pre-match value.(Nielsen et al., 2004).Th e aforementioned values are high enough to depolarize the muscle membrane potential and reduce force development markedly (Cairns & Dulhunty, 1995).Takarada (2003) found that plasma K + concentrations had signifi cantly increased aft er the match, whereas plasma Na + concentration gradually decreased.Interestingly, we demonstrated reduced levels of plasma [K + ] aft er a soccer match.However, the plasma [Na + ] concentration was unchanged aft er an offi cial soccer match in U-21 players, which is in line with Takarada (2003).Fraser et al. (2002) observed reduced maximal activity of the Na + /K + pump following diff erent types of exercise.While these plasma values do not provide a clear picture of the concentrations around the contracting muscle fi bres in soccer (Bangsbo, Mohr, & Krustrup, 2006), further research is needed to reveal what may cause fatigue during a soccer game.
Th is study approached the analysis of muscle damage markers before and immediately aft er the match.Th e strength of our study lies in the fact that the results come from an offi cial soccer match played by U-21 professional players.We can say that most selected markers of muscle damage were infl uenced by a soccer match.However, the results of this study, along with those of other numerous studies are inconsistent because of the infl uence of type, duration and intensity of exercise.Moreover, some markers show great variability among individuals.Th erefore, more studies like this are needed to reveal which markers are essential for designing the proper training programme and how to prevent injuries and overtraining in professional soccer players.

TABLE 2
Electrolyte values of the players before and after the played match.Data are presented as Mean ± SD