Physical Fitness and Performance

Muscle and Blood Metabolites during a Soccer Game: Implications for Sprint Performance PETER KRUSTRUP1, MAGNI MOHR1, ADAM STEENSBERG2, JESPER BENCKE3, MICHAEL KJÆR4, and JENS BANGSBO1 1 Institute of Exercise and Sport Sciences, Department of Human Physiology, University of Copenhagen, Copenhagen, DENMARK; 2Copenhagen Muscle Research Centre, Rigshospitalet, Department of Infectious Diseases, Copenhagen, DENMARK; 3Department of Orthopedic Surgery, Hvidovre University Hospital, Copenhagen, DENMARK; and 4Sports Medicine Research Unit, Copenhagen University Hospital, Copenhagen, DENMARK

ABSTRACT KRUSTRUP, P., M. MOHR, A. STEENSBERG, J. BENCKE, M. KJÆR, and J. BANGSBO. Muscle and Blood Metabolites during a Soccer Game: Implications for Sprint Performance. Med. Sci. Sports Exerc.,Vol. 38, No. 6, pp. 1165 – 1174, 2006. Purpose: To examine muscle and blood metabolites during soccer match play and relate it to possible changes in sprint performance. Methods: Thirty-one Danish fourth division players took part in three friendly games. Blood samples were collected frequently during the game, and muscle biopsies were taken before and after the game as well as immediately after an intense period in each half. The players performed five 30-m sprints interspersed by 25-s recovery periods before the game and immediately after each half (N = 11) or after an intense exercise period in each half (N = 20). Results: Muscle lactate was 15.9 T 1.9 and 16.9 T 2.3 mmolIkgj1 d.w. during the first and second halves, respectively, with blood lactate being 6.0 T 0.4 and 5.0 T 0.4 mM, respectively. Muscle lactate was not correlated with blood lactate (r2 = 0.06 – 0.25, P > 0.05). Muscle glycogen decreased (P G 0.05) from 449 T 23 to 255 T 22 mmolIkgj1 d.w. during the game, with 47 T 7% of the muscle fibers being completely or almost empty of glycogen after the game. Blood glucose remained elevated during the game, whereas plasma FFA increased (P G 0.05) from 0.45 T 0.05 to 1.37 T 0.23 mM. Mean sprint time was unaltered after the first half, but longer (P G 0.05) after the game (2.8 T 0.7%) as well as after intense periods in the first (1.6 T 0.6%) and second halves (3.6 T 0.5%). The decline in sprint performance during the game was not correlated with muscle lactate, muscle pH, or total glycogen content. Conclusion: Sprint performance is reduced both temporarily during a game and at the end of a soccer game. The latter finding may be explained by low glycogen levels in individual muscle fibers. Blood lactate is a poor indicator of muscle lactate during soccer match play. Key Words: INTERMITTENT EXERCISE, FATIGUE, BLOOD LACTATE, MUSCLE LACTATE, GLYCOGEN DEPLETION

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occer is an intermittent sport in which the aerobic energy system is highly taxed with average and peak heart rates around 85 and 98% of maximal values, respectively (2,21,24). The observation that elite soccer players perform 150 – 250 brief, intense actions during a game (19) and have blood lactate values of 2 –14 mM (3,10,25) indicates that the rate of anaerobic energy

turnover is high during periods of a game. However, it is still uncertain how well blood lactate measurements reflect the muscular lactate concentration. To achieve a better understanding of the anaerobic energy turnover during soccer match play, direct measurements of muscle lactate and other metabolites are therefore required. Recent findings using computerized time –motion analysis of elite male and female soccer players have indicated that the players are fatigued temporarily during a game (19). Thus, in the 5-min period following the most intense period of the match, the amount of high-intensity exercise was reduced to levels below game average. Therefore, it would be of interest to examine whether, and to what extent, sprint performance is impaired after an intense exercise period during a game, and to relate any alterations to changes in muscle variables that have been suggested to cause fatigue during intense exercise such as lactate and pH (27).

Address for correspondence: Jens Bangsbo, Institute of Exercise and Sport Sciences, August Krogh Institute, Universitetsparken 13, DK-2100 Copenhagen K, Denmark; E-mail: [email protected]. Submitted for publication February 2005. Accepted for publication November 2005. 0195-9131/06/3806-1165/0 MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ Copyright Ó 2006 by the American College of Sports Medicine DOI: 10.1249/01.mss.0000222845.89262.cd

1165 Copyright @ 2006 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

Several studies have provided evidence that the ability to perform high-intensity exercise is reduced towards the end of elite as well as subelite soccer games (19,21,23,26). Thus, it has been observed that the amount of highintensity running is reduced in the last 15-min period of top-class soccer (19,26) and that jump, sprint, and intermittent exercise performance is lowered after, compared with before, a soccer game (21,23). However, the underlying mechanism behind a reduced exercise performance at the end of soccer games is unclear. One candidate is depletion of glycogen stores because some (28), but not all (14,29) investigations have observed that muscle glycogen decreases to levels below the required value to maintain maximal glycolytic rate (~200 mmolIkgj1 d.w.) (4). To further elucidate the role of muscle glycogen in the fatigue development at the end of soccer matches, muscle biopsies can be collected after a soccer game for analysis of the glycogen content of the individual muscle fibers. It would also be of value to investigate how a progressive depletion of the glycogen stores is related to changes in blood variables such as FFA and glucose. Thus, the aim of the present study was to examine muscle and blood metabolites during soccer match play and to relate those to sprint performance during and after the game. An additional purpose was to investigate how blood lactate reflects muscle lactate concentration during a soccer match.

MATERIAL AND METHODS Subjects. Thirty-one fourth division Danish soccer players, with a mean age of 28 yr (range: 21 – 33), an average height of 179 cm (173 – 191), and an average body mass of 75.7 kg (63.7 – 83.6) participated in the study. The players were fully informed of experimental procedures and possible discomforts associated with the study before giving their written informed consent to participate. The study was approved by the ethics committee of the Copenhagen and Frederiksberg communities. Experimental design/protocol. The players took part in three friendly games. A number of physiological measurements were performed at fixed times throughout the game as well as after intense exercise periods within each half. Eleven players had blood samples taken frequently during the match—at rest, before the game, after 5, 15, and 45 min of each half, and 15 min after the game. These players had biopsies taken before and after the game. Another 20 players had blood samples taken before and after each half as well as after an intense exercise period in each of the two halves. For fourteen of these players, a muscle biopsy was taken after an intense exercise period in each half, which included fast running (>18 kmIhj1) and sprinting (>25 kmIhj1). All players had heart rate measured throughout the game. To evaluate sprint performance during match play, a repeated sprint test was performed before the game as well as immediately 1166

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after each half (N = 11) or immediately after an intense period within each half (N = 20). Preparation procedures. Prior to the matches, the players refrained from strenuous exercise and intake of alcohol for 48 h and from tobacco and caffeine for 12 h. The games started early in the afternoon (2 – 4 p.m.). The diet was controlled 16 h prior to the game and included 200 g of meat, 100 g of vegetables, and 300 g of pasta the night before the game; 100 g of cereals with milk in the morning; and a light meal of bread and fruit 2 h before kickoff. The players arrived at the stadium 1.5 h before the games. Before the warm-up a catheter (18 g, 32 mm) was placed in an antecubital vein and covered by a wrist bandage. In preparation for later obtainment of biopsies in m. vastus lateralis an incision was made under local anesthesia (20 mgImLj1 lidocain without adrenalin) and covered by sterile band aid strips and a thigh bandage. In addition, a heart rate monitor was placed on each subject (see below). Muscle and blood sampling. The muscle biopsies (~70 – 120 mg w.w.) were obtained from m. vastus lateralis in the right leg using the needle biopsy technique with suction. All biopsies were obtained with the subjects lying in the supine position on one of four beds standing 2 m from the sideline. Muscle biopsies were collected 15 – 30 s after cessation of match play. Blood samples were taken from an arm vein in the right arm using 5-mL syringes. Some of the blood samples were taken simultaneously with muscle samples, with the subjects lying on a bed. The remaining blood samples were collected within 30 s of match play, with the subjects sitting on a bed positioned 2 m from the sideline. Heart rate measurements. Heart rate was recorded in 5-s intervals during the entire game using a Polar Vantage NV heart rate monitor (Polar Electro Oy, Kempele, Finland). The monitor consisted of a chest belt and wrist receiver (weighing approximately 100 g). The data was subsequently stored on a personal computer using a Vantage Interface (Polar Electro Oy, Kempele, Finland). Repeated sprint test. All players performed one repeated sprint test before and two during the game. Each repeated sprint test consisted of five 30-m sprints, separated by a 25-s period of active recovery, during which the players jogged back to the starting line. Thus, each test lasted approximately 2 min. The test was started 20 – 30 s after obtainment of a muscle biopsy. Thus, the total time delay from match play to the sprint test was 35 – 60 s. The sprint times were recorded by infrared light sensors with a precision of 0.01 s (Time It, Eleiko Sport, Halmstad, Sweden). Muscle analysis. The muscle tissue was immediately frozen in liquid N2 and stored at j80-C. The frozen sample was weighed before and after freeze drying to determine water content. After freeze drying, the muscle samples were dissected free of blood, fat, and connective tissue, and about 1 mg d.w. tissue was extracted in a solution of 0.6 M perchloric acid (PCA) and 1 mM EDTA, neutralized to pH 7.0 with 2.2 M KHCO3 and stored at j80-C until analyzed for lactate and CP by a flurometric http://www.acsm-msse.org

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TABLE 1. Muscle water content, ATP, IMP, CP, lactate, pH, and glycogen before and after a soccer match as well as before and after intense exercise periods in the first and second halves.

Water content (%) Muscle ATP (mmolIkgj1 d.w.) Muscle IMP (mmolIkgj1 d.w.) Muscle CP (mmolIkgj1 d.w.) Muscle lactate (mmolIkgj1 d.w.) Muscle H+ (nmolIkgj1 d.w.) Muscle pH (blog H+) Muscle glycogen (mmolIkgj1 d.w.)

Before Match

After Match

After an Intense Period in the First Half

After an Intense Period in the Second Half

76.0 T 0.4 (74.4 – 77.3) 26.4 T 2.3 (24.1 – 28.8) 0.0 T 0.0 (0.0 – 0.0) 88 T 2 (74 – 98) 4.2 T 0.5 (2.7 – 6.1) 57 T 2 (54 – 68) 7.24 T 0.02 (7.17 – 7.30) 449 T 23 (387 – 626)

78.2 T 0.7* (75.7 – 81.1) 23.0 T 0.4* (22.6 – 23.5) 0.3 T 0.1 (0.0 – 0.6) 79 T 3* (68 – 95) 13.0 T 1.8* (8.7 – 23.9) 69 T 2 (60 – 72) 7.17 T 0.01 (7.14 – 7.22) 255 T 22* (168 – 375)

77.6 T 0.2* (76.7 – 79.0) 25.6 T 0.2 (25.3 – 25.9) 0.2 T 0.1 (0.0 – 0.5) 76 T 3* (58 – 93) 15.9 T 1.9* (6.4 – 29.3) 111 T 9* (81 – 159) 6.96 T 0.03* (6.80 – 7.09) 296 T 23* (199 – 403)

78.8 T 0.3*# (77.7 – 80.2) 22.6 T 1.0*# (19.1 – 26.8) 0.6 T 0.2* (0.0 – 1.1) 67 T 3*# (44 – 85) 16.9 T 2.3* (2.6 – 36.4) 86 T 4*# (71 – 110) 7.07 T 0.02*# (6.96 – 7.15) 241 T 16*# (161 – 319)

Values are means T SEM. Pre- and postmatch water content and glycogen (N = 11); ATP, IMP, CP, and lactate (N = 7); H+ and pH (N = 5). Intense exercise water content, CP, lactate, and glycogen (N = 14); ATP, IMP, H+ , and pH (N = 8). * Significantly different (P G 0.05) from before match; # significantly different from after an intense exercise period in the first half.

assay (17). Muscle pH was measured by a small glass electrode (Radiometer GK2801, Copenhagen, Denmark) after homogenizing a freeze-dried muscle sample of about 2 mg d.w. in a nonbuffering solution containing 145 mM KCl, 10 mM NaCl, and 5 mM iodoacetic acid. Another 2 mg d.w. of muscle tissue were extracted in 1M PCA, neutralized to pH 8.0 with 2M KOH, and stored at j80-C until analyzed for adenine nucleotides (ATP, ADP, AMP and IMP), using reverse phase high-performance liquid chromatography (HPLC) (13). In addition, 1– 2 mg d.w. muscle tissue was extracted in 1M HCl and hydrolyzed at 100-C for 3 h, and the glycogen content was determined by the hexokinase method (17). A part of each biopsy obtained before and after the game was mounted in an embedded medium (OCT Compound Tissue-Tek, Sakura Finetek, Zoeterwoude, The Netherlands) and frozen in isopentane that was cooled to the freezing point in liquid nitrogen. These samples were stored at j80-C until analyzed for fiber-type distribution and fiber-type –specific glycogen content by histochemical analysis. Five serial 10-Km-thick sections were cut at j20-C and incubated for myofibrillar adenosine triphosphate (ATPase) reactions at pH 9.4, after preincubation at pH 4.3, 4.6, and 10.3. Based on the myofibrillar ATP staining, individual fibers were classified under light microscopy as ST, FTa, or FTx. To evaluate the relative glycogen content of individual fibers, one 16-Km-thick transverse section was cut at j20-C and stained for glycogen by the periodic acid– Schiff (PAS) reaction. Under light microscopy, the staining intensity of the fibers was rated as full, partly full, almost empty, or empty, and ranked from 3 to 0 (15). Blood analysis. Within 10 s of sampling, 100 KL of blood was hemolyzed in an ice-cold 100-KL Triton X-100 buffer solution, and was later analyzed for lactate and glucose using an YSI 2300 lactate analyzer (Yellow Spring Instruments, Yellow Springs, OH). The rest of the sample was rapidly centrifuged for 30 s. From this, plasma was collected and stored at j20-C until subsequent analysis. MUSCLE METABOLITES AND FATIGUE IN SOCCER

Plasma potassium concentration was measured using a flame photometer (Radiometer FLM3), with lithium as the internal standard. Plasma glycerol was determined on an automatic analyzer (Cobas Fara, Roche, France). Plasma free fatty acid (FFA) was measured fluorometrically using an enzymatic kit (WAKO Chemical, Germany), and plasma ammonia (NH3) was determined spectophotometrically (13). Fluid loss and intake. To determine sweat loss during the game, the players were weighed wearing dry shorts immediately before the match, at half time, and immediately after the match using a digital weight (OHAUS 1–10, New Jersey). The players were allowed to drink water ad libitum during the game, and their water intake was recorded. Statistics. Changes in performance of repeated sprints during the game were evaluated by two-way analysis of variance (ANOVA) with repeated measures. Changes in blood metabolites before and during the game were determined by a one-way ANOVA with repeated measures. When a significant interaction was detected, data were subsequently analyzed using a Newman – Keuls post hoc test. Differences in muscle metabolites between rest and match play were determined by Student’s unpaired t-test, whereas differences between the two halves were determined by Student’s paired t-test. Differences in ratings of fiber-type – specific glycogen content between rest and after the game were evaluated by a Wilcoxon signed rank test. Correlation coefficients were determined and tested for significance using the Pearson’s regression test. A significance level of 0.05 was chosen. Data are presented as means T standard error of the mean (SEM).

RESULTS Heart Rate during Match Play Mean heart rate during the match was 156 T 13 (T SD) bpm, and peak heart rate was 187 T 9 bpm. No significant difference was observed in mean heart rate between the first and the Medicine & Science in Sports & Exercised

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FIGURE 1—Muscle lactate (A), pH (B), and glycogen (C) before and after a soccer game as well as after intense periods in the first and second halves. Individual values are presented.

second halves (157 T 15 vs 155 T 13 bpm), whereas peak heart rate reached in the first half was higher (P G 0.05) than in the second half (186 T 9 vs 181 T 10 bpm). Muscle Metabolites and pH during Match Play Muscle metabolite concentrations, water content, and pH before, during, and after a soccer game are presented in Table 1. Muscle adenosine nucleotides and CP. Muscle ATP was 22.6 T 1.0 (T SEM) mmolIkgj1 d.w. after an intense period in the second half, which was lower (P G 0.05) than at rest (26.4 T 2.3 mmolIkgj1 d.w.). Muscle IMP was 0.6 T 0.2 mmolIkgj1 d.w. after an intense period in the second half, which was higher (P G 0.05) than at rest. Muscle CP was 67 T 3 mmolIkgj1 d.w. after an intense period in the second half, which was lower (P G 0.05) than at rest (88 T 2 mmolIkgj1 d.w.) and during the first half (76 T 3 mmolIkgj1 d.w.). Muscle lactate and pH. Muscle lactate after intense periods in the first and second halves was 15.9 T 1.9 and 16.9 T 2.3 mmolIkgj1 d.w., respectively, which was about fourfold higher (P G 0.05) than at rest (Fig. 1A). Large intraindividual variations were found in muscle lactate 1168

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between the two halves (j14.4 to 27.8 mmolIkgj1 d.w.), with a CV value of 74%. Muscle pH was 6.96 T 0.03 after an intense period in the first half, which was lower (P G 0.05) than after an intense period in the second half (7.07 T 0.02) and at rest (7.24 T 0.02) (Fig. 1B). The corresponding

FIGURE 2—Relative glycogen content in ST, FTa, and FTx fibers as well as all fibers before and immediately after a soccer match. Values are means (N = 10). http://www.acsm-msse.org

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TABLE 2. Blood lactate and glucose as well as plasma FFA, glycerol, NH3, K+, and insulin concentrations at rest, after the first half, before and after the second half, and after intense exercise periods in the first and second halves (N = 20).

Blood lactate (mM) Blood glucose (mM) Plasma FFA (KM) Plasma glycerol (KM) Plasma NH3 (KM) Plasma K+ (mM) Plasma insulin (KM)

At rest

During First Half

After First Half

Before Second Half

During Second Half

After Second Half

1.3 T 0.1 (0.7 – 2.2) 4.5 T 0.1 (3.2 – 5.8) 390 T 57 (174 – 1008) 81 T 29 (35 – 136) 59 T 7 (27 – 136) 3.9 T 0.0 (3.6 – 4.3) 15.2 T 2.0 (6.3 T 34.0)

6.0 T 0.4* (3.8 – 9.6) 6.1 T 0.3* (4.0 – 8.2) 555 T 74 (186 – 1265) 185 T 29* (97 – 251) 203 T 16* (97 – 310) 4.9 T 0.1* (4.4 – 5.5) 7.4 T 0.5 (5.3 T 11.3)

4.1 T 0.4* (1.3 – 6.0) 5.4 T 0.4* (2.8 – 8.9) 671 T 95* (185 – 1693) —

2.1 T 0.2* (0.9 – 3.5) 4.1 T 0.2* (2.9 – 5.6) 1066 T 193* (248 – 2845) —

3.9 T 0.4* (2.1 – 5.9) 4.9 T 0.2* (4.0 – 6.3) 1365 T 111* (831 – 2276) —

193 T 19* (78 – 283) 4.6 T 0.1* (3.6 – 5.4) —

128 T 15* (50 – 240) 4.2 T 0.1 3.9 – 4.6 —

5.0 T 0.4*# (2.3 – 8.6) 5.3 T 0.3*# (4.0 – 6.6) 740 T 75*# (401 – 1417) 234 T 40*# (82 – 310) 217 T 20* (68 – 342) 4.8 T 0.1 4.2 – 5.6 6.0 T 0.9* 3.0 T 11.9

Values are means T SEM. * Significantly different (P G 0.05) from before match.

#

199 T 21* (71 – 350) 4.3 T 0.1 3.9 – 5.0 —

Significantly different from after an intense period in the first half.

muscle H+ concentrations were 111 T 9, 86 T 4, and 57 T 2 nmolILj1, respectively. Muscle glycogen. Muscle glycogen was 449 T 23 mmolIkgj1 d.w. at rest and was 42 T 6% lower (P G 0.05) after the game (Fig. 1C). The 10 subjects that were analyzed for fiber-type –specific glycogen depletion during

the game had 58.5 T 3.5% ST fibers, 26.9 T 2.6% FTa fibers, and 14.6 T 3.0% FTx fibers. Before the game, 73 T 6% of all fibers were rated as full with glycogen, whereas this value was lower (P G 0.05) after the game (19 T 4%, Fig. 2). After the game, a total of 36 T 6% of the individual muscle fibers were almost empty, and another 11 T 3%

FIGURE 3—Blood lactate (A), plasma FFA (B), and plasma NH3 (C) concentrations before, during, and after a soccer game. Data are means T SEM (N = 11) as well as individual values. MUSCLE METABOLITES AND FATIGUE IN SOCCER

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after an intense period in the first and second halves, respectively, which was higher (P G 0.05) than at rest (3.9 T 0.0 mmolILj1) (Table 2). Peak plasma NH3 and K+ reached during the game was 283 (168 – 362) KmolILj1 and 5.1 (4.5 –5.6) mmolILj1, respectively. Fluid Loss and Intake The weight loss during a game was 0.84 T 0.14 (0.31 – 1.48) L, or 1.1 T 0.2 (0.4 – 2.1) % of the body mass. The fluid intake was 0.69 T 0.08 (0.35 –1.05) L. Thus, the total fluid loss during the game was 1.53 T 0.17 (1.10 –2.16) L, corresponding to 2.0 T 0.2 (1.3 – 2.8) % of the body mass. Repeated Sprint Performance during Match Play FIGURE 4—Individual relationship between muscle and blood lactate concentration after intense exercise periods in the first half and during a soccer match. The filled squares and open squares represent measurements performed in the first (r2 = 0.25, P >0.05) and second halves (r2 = 0.06, P > 0.05), respectively.

The mean time for five 30-m sprints was 4.72 T 0.05 s after the game, which was 2.8 T 0.7% longer (P G 0.05)

were completely empty of glycogen (Fig. 2). A total of 54 T 10 and 46 T 11% of the ST and FTa fibers, respectively, were completely or almost empty of glycogen after the game, whereas this was the case for 25 T 10% of the FTx fibers (Fig. 2). Blood Variables during Match Play Blood lactate and glucose. Blood lactate was 0.9 T 0.2 mmolILj1 at rest and increased (P G 0.05) to 6.7 T 0.9 mmolIL-1 after 5 min of play, which was higher (P G 0.05) than at the end of the game (Fig. 3A). Peak lactate reached during the game was 7.9 T 0.7 (4.2 – 11.9) mmolILj1. Blood lactate after intense periods in the first and second halves was 6.0 T 0.4 and 5.0 T 0.4 mmolILj1, respectively (Table 2). No relationship was observed between muscle and blood lactate concentration for the first or the second halves (r2 = 0.25 and 0.06, respectively, P > 0.05) (Fig. 4). Blood glucose was 4.3 T 0.1 mmolILj1 at rest and higher (P G 0.05) after 5 min of play (5.2 T 0.2 mmolILj1), after which it remained unaltered throughout the game (90 min: 4.9 T 0.4 mmolILj1). Plasma FFA, glycerol, and insulin. Plasma FFA was 433 T 77 KmolILj1 at rest and about 1.5- and 3-fold higher (P G 0.05) after the first and second halves, respectively (Fig. 3B). Plasma glycerol was 185 T 28 and 234 T 40 KmolILj1 after intense periods in the first and second halves, respectively, which was higher (P G 0.05) than at rest (81 T 29 KmolILj1) (Table 2). Plasma insulin was 11.8 T 1.5 KmolILj1 at rest and was lower (P G 0.05) after the first and second halves (7.3 T 0.8 and 5.2 T 0.6 KmolILj1, respectively). Plasma NH 3 and K + . Plasma NH 3 was 38 T 6 KmolILj1 at rest and about sixfold higher (P G 0.05) after 5 min of play, after which it remained unaltered (Fig. 3C). Plasma NH3 was 203 T 16 and 217 T 20 KmolILj1 after an intense period in the first and second halves, respectively (Table 2). Plasma K+ was 4.9 T 0.1 and 4.8 T 0.1 mmolILj1 1170

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FIGURE 5—Time of five 30-m sprints before the game (filled circles), after the first half (open circles), and after the game (filled triangles) (A, N = 11) as well as time of five 30-m sprints before the game (filled circles) and after intense exercise periods during the first (open circles) and second halves (filled triangles) (B, N = 20). The sprints were separated by 25-s periods of active recovery. Data are means T SEM. http://www.acsm-msse.org

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FIGURE 6—Individual relationship between muscle lactate and the decrease in mean sprint time of five 30-m sprints performed before and during the game. The filled squares and open squares represent data obtained during the first (r2 = 0.14, P > 0.05) and second halves (r2 = 0.13, P > 0.05), respectively.

than before the game. All five sprints were slower (P G 0.05) after compared with before the game (Fig. 5A). No difference was observed in mean sprint time after the first half compared with before the game (4.60 T 0.05 and 4.61 T 0.06 s, respectively) (Fig. 5A). After intense periods in the first and second halves, the mean sprint time was 1.6 T 0.6 and 3.6 T 0.5% longer (P G 0.05) than before the game (4.72 T 0.07 and 4.80 T 0.06 vs 4.64 T 0.05 s) (Fig. 5b). The third, fourth, and fifth sprints performed after an intense period in the first half and all five sprints performed after an intense period in the second half were slower (P G 0.05) compared with before the game (Fig. 5B). Relationship between Muscle and Blood Metabolites and Sprint Performance No interindividual correlation was observed between muscle lactate and the decrease in performance of repeated sprints after intense exercise periods in the first half (r2 = 0.14, P > 0.05) or the second half (r2 = 0.13, P > 0.05) (Fig. 6). Moreover, the decrease in performance of repeated sprints after intense exercise periods in the first and second halves were not correlated to muscle pH (r2 = 0.00 and 0.12, respectively, P > 0.05), total muscle glycogen (r2 = 0.02 and 0.01, P > 0.05), muscle ATP (r2 = 0.05 and 0.07, P > 0.05), or muscle IMP (r2 = 0.01 and 0.03, P > 0.05). No relationship was found between the decrease in sprint performance during the game and any of the measured blood metabolites.

DISCUSSION The present study provides information about the changes in performance and alterations in muscle and blood metabolites during a soccer match. It was shown that sprint performance was reduced both temporarily during and at the end of a match. Muscle lactate and H+ were only moderately elevated during the game and did not correlate MUSCLE METABOLITES AND FATIGUE IN SOCCER

with the lower performance during the match. At the end of the game about half of the muscle fibers were almost empty or empty of glycogen, which may contribute to the reduced performance at the end of a match. A lack of correlation was observed between muscle and blood lactate, indicating that caution should be taken when interpretating blood lactate levels obtained during a soccer match. Blood glucose was maintained at elevated levels throughout the match, whereas there was a progressive increase in the FFA concentration. Intensity of the examined matches. The matches examined in the present study were friendly games played by Danish fourth division players, and it should be considered how well they represent games played at an elite level. The mean and peak heart rate was observed to be 156 and 187 bpm, respectively, which is 5 –10 bpm lower than values reported for elite soccer (4,10), whereas average blood lactate values of 5 mmolILj1 were of the same magnitude. For seven of the biopsied players, time – motion analyses were also performed. These analyses showed that the total distance covered (9.75 T 0.33 km) and total amount of high-intensity running (1.62 T 0.12 km) were 10 and 20% lower, respectively, than those observed at an elite level (3,19). It was furthermore observed that the amount of high-intensity running in a 5-min period prior to muscle sampling was 145 T 23 m, which is 25% less than in the most intense 5-min period for elite soccer players (19). Nevertheless, the reduction in sprint performance after the game was similar to values seen for elite players (23). Together, these findings suggest that the absolute intensity in the investigated games was somewhat lower than for elite competitive games, and that the relative physiological strain of the players was comparable for players at an elite level. Temporary fatigue during a soccer match. In a study of competitive elite soccer, it has been observed that the amount of high-intensity running decreases below game average after the most intense 5-min period of the game, suggesting that temporary fatigue occurs during match play (19). The present data show that performance of the third, fourth, and fifth sprints carried out after a period of intense exercise during the first half was reduced compared with before the game. This finding together with the observation that sprint performance at the end of the first half was the same as before the game, provides direct evidence that fatigue occurs temporarily during a game. It was furthermore observed that performance of all five sprints was reduced after an intense exercise period in the second half. Several mechanisms have been suggested to cause fatigue during intense exercise, including inhibitory effects of muscle lactate and pH (27). In the present study, muscle biopsies were collected immediately after cessation of an intense exercise period (15 – 30 s) and the sprint test was initiated shortly after the sampling (20 –30 s). As the recovery processes are slow for muscle lactate, glycogen and ATP (G 0.1 mmolIkgj1 d.w. sj1), and muscle H+ (G 0.1 nmolILj1Isj1) after intense exercise (6,7), the data Medicine & Science in Sports & Exercised

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obtained for these variables are likely to represent the values during the game and immediately before the sprint test. It is therefore possible to correlate these variables to the reduction in sprint performance. After intense periods of exercise in the two halves, we observed moderate changes in muscle lactate amounting to about one fourth of the changes observed during exhaustive intermittent running (15), and no significant relationship was observed between muscle lactate and the reduction in sprint performance (r2 = 0.13 – 0.14; Fig. 6). In accordance with several other studies (4,15,20), these data suggest that accumulation of muscle lactate is not the cause of fatigue during intense exercise. Similarly, muscle pH was only moderately reduced and no relationship was found to the lowered performance, indicating that lowered pH was not the cause of fatigue during the game. This is in accordance with findings in a number of other studies using intense intermittent exercise (15,20). The CP concentration was also measured in the collected biopsies, and average values were observed to be approximately 20 mmolIkgj1 d.w. lower after an intense period in the first half compared with at rest. Because the rate of muscle CP resynthesis has been reported to be as high as 0.5 mmolIkgj1 d.w. sj1 after intense exercise and differs between individuals (6,7), it is likely that the reported values significantly underestimate the CP breakdown during match play, and it is not of value to correlate the obtained muscle CP values with the reduced sprint performance. In another study using intense intermittent exercise (the yo-yo intermittent recovery test), it was observed that subjects were able to continue this type of exercise with rather low concentrations of CP and that they had no change in muscle CP in the last phase of the exhaustive exercise (15). Based on this finding and the observations in a number of studies using intense kneeextensor exercise (4,13,20,22), it may be suggested that fatigue during the game was not caused by low CP levels. During the match the muscle IMP concentrations were higher compared with before the game, and the elevated blood NH3 levels also indicate that the AMP deaminase reaction was significantly stimulated. On the other hand, the muscle IMP levels were considerably lower than those observed during exhaustive exercise (13), and ATP was only moderately reduced. Thus, it is not likely that fatigue occurred as a result of a low energy status of the contracting muscles. Fatigue occurring during intense exercise periods has been suggested to be related to accumulation of potassium in muscle interstitium (20,22). In the present study the plasma potassium concentration was observed to be 5 mM, with individual values above 5.5 mM, which is only slightly lower than the values observed 30 – 60 s after exhaustive incremental intermittent exercise (15). Thus, the hypothesis that accumulation of potassium in muscle interstitium is involved in a temporary reduction in force development after an intense exercise period during a soccer game should be explored (22). Nevertheless, it should be emphasized that the cause of temporary fatigue is likely to be multifactorial. 1172

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Fatigue towards the end of a soccer match. Immediately after the game, sprint performance was also reduced. Development of fatigue during prolonged intermittent exercise has been associated with lack of muscle glycogen, and it has been demonstrated that elevating muscle glycogen prior to exercise through a carbohydrate diet elevates performance during such a type of exercise (1,4). In a number of studies muscle glycogen has been determined before, during, and after a game. Saltin (28) observed that the muscle glycogen stores were almost depleted at half time, when the prematch levels were low (~200 mmolIkgj1 d.w.). In that study, some players started the game with normal muscle glycogen levels (~400 mmolIkgj1 d.w.), and the values were still rather high at half time, but below 50 mmolIkgj1 d.w. at the end of the game. Others have found the concentrations to be approximately 200 mmolIkgj1 d.w. after the game (14,29), indicating that muscle glycogen stores are not always depleted in a soccer game. In the present study the muscle glycogen concentration at the end of the game was reduced to 150 – 350 mmolIkgj1 d.w. Thus, there was still glycogen available, but the histochemical analysis revealed that about half of the individual muscle fibers of both types were depleted or almost depleted for glycogen. Therefore, it is possible that such a depletion of glycogen in some fibers does not allow for a maximal effort in single and repeated sprints. Factors such as dehydration and hyperthermia may also contribute to the fatigue development in the latter period of a soccer game (18,25). Soccer players have been reported to lose up to 3 L of fluid during games in a normal thermal environment and as much as 4 –5 L in a hot and humid environment (25), and it has been observed that 5- and 10-m sprint times are slowed by hypohydration amounting to 2.7% of the body weight (18). However, in the present study, the fluid loss of the players was about 1% of the body weight, and no effect on core or muscle temperature was observed in a study with a similar loss of fluid (21). Thus, it appears that the fluid loss was not an important component in our observation of a reduced ability to perform repeated sprints at the end of the game. Relationship between muscle and blood lactate. An interesting finding in the present study was a lack of correlation between muscle and blood lactate (Fig. 4). Similarly, a scattered relationship with a low correlation coefficient has been observed between muscle and blood lactate when subjects performed repeated intense exercise carrying out the yo-yo intermittent recovery test ((15); Fig. 7). This is in contrast to continuous exercise where the blood lactate concentrations are lower but reflect well the muscle lactate concentrations during exercise ((16); Fig. 7). These differences between intermittent and continuous exercise are probably due to different turnover rates of muscle and blood lactate during the two types of exercise. In general, the muscle lactate concentration is a product of the production and the removal of lactate either as turnover within the muscle or release to the blood stream, which is restricted during intense exercise (5). The blood lactate concentration http://www.acsm-msse.org

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FIGURE 7—Individual relationship between muscle lactate, expressed in millimoles per litter of fluid, and blood lactate, during a soccer match (filled circles; data from the present study), at exhaustion in the yo-yo intermittent level 1 recovery test (filled triangles; data from (15)) as well as after 20 min of continuous cycle exercise at 80% of maximal oxygen uptake (open circles; data from (16)).

is the result of the release of lactate from the contracting muscles and the clearance from the blood, which is influenced by the metabolism in various tissues such as the heart and liver (8). To illustrate the difference between blood and muscle lactate turnover in soccer, data from a study using low-intensity exercise after intense exercise can be used (5). The net rate of clearance of lactate from the blood can be estimated to be 0.25 mmolILj1Iminj1 at a blood lactate concentration of 3–5 mmolILj1, which was in a range observed during the first half in the present study. At the same time, the mean muscle lactate concentration was about 8 or 10 mmolILj1, and the release of lactate to the blood was about 0.5 mmolIkgj1Iminj1 (5). Furthermore, the rate of decrease in muscle lactate was around 1.8 mmolILj1Iminj1, or about sevenfold higher than the rate of decrease in blood lactate. This means that during intermittent exercise, the blood lactate levels can be high even though the muscle lactate concentration is relative low. It may also be that the closer the high-intensity exercise is performed in relation to the sampling, the larger the difference will be between the muscle and blood lactate concentrations. Thus, muscle and blood lactate are not only influenced by the activities prior to the sampling, but also when the high-intensity activity is performed. This can explain why no correlation was observed between the high-intensity running 5 min prior to sampling and the blood lactate concentrations (r2 = 0.07, N = 10, P > 0.05). To summarize, because the clearance rate of blood lactate is lower than the turnover rate of lactate in the muscle, the blood lactate concentration represents an accumulated response of the lactate production in the muscles during periods in the game with repeated intense exercise. Thus, the rather high blood lactate concentration often seen in soccer (2,10) may not represent a high lactate production in a single

action during the game. Nevertheless, the finding of high blood lactate and moderate muscle lactate concentrations in the present study suggest that the rate of glycolysis is high for short periods of time during a game. Metabolism during a soccer match. In the present study it was observed that the FFA concentration in the blood increased during the game, and more so during the second half. The frequent rest and low-intensity periods of the game would have allowed for a significant blood flow to adipose tissue, which promotes release of FA. This effect is also illustrated by the high FFA concentration at half time and after the game. A high rate of lipolysis is also supported by the elevated levels of glycerol, even though the increases were smaller than during continuous exercise, which probably does reflect a high turnover of glycerol, for instance, as a gluconeogenic precursor in the liver (2). Hormonal changes may have caused the progressive increase in the FFA level. The insulin concentrations were lowered and catecholamines levels were probably elevated during the match (2), stimulating a high rate of lipolysis, and thus release of FA to the blood (11). These effects are counteracted by high levels of lactate, which suppresses mobilization of FA from the adipose tissue (9,11). Therefore, it is likely that the lowered insulin and lactate concentrations as well as higher levels of catecholamines progressively increased the FFA concentrations towards the end of the game. These changes in FFA during the match may have caused a higher uptake and oxidation of FFA by the exercising muscles (30). In addition, a higher utilization of muscle TG might have occurred in the second half due to elevated catecholamine concentrations (12). Both processes may have been compensating for the progressive lowering of muscle glycogen and maintained the blood glucose concentration high. Summary. The present study has demonstrated that sprint performance is reduced temporarily both during and towards the end of a soccer game. It is unclear what causes the development of fatigue during the game, whereas fatigue towards the end of the game may be associated with reduced glycogen levels in individual muscle fibers. The blood lactate concentrations do reflect the production of lactate in the contracting muscles during a soccer match, but such values should be interpreted carefully. We would like to thank the soccer players involved in the study for their committed participation. The excellent technical assistance and skilful involvement of Ingelise Kring, Merete Vannby, Winnie Taagerup, Lise Svendsen, Birgitte Rejkjær Krustrup, Charlotte Barfred, Inge Krustrup, Jesper Jansson, and Helga Ellingsgaard is appreciated. We would also like to acknowledge Thore Hillig, Jens Jung Nielsen, Christoffer Krustrup, Jane Kjølhede, and Lene Kjølhede for practical assistance. The study was supported by Team Denmark and The Sports Research Council (Idrættens Forskningsra˚d).

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