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ARTICLE

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Effect of a carbohydrate-protein multi-ingredient supplement on intermittent sprint performance and muscle damage in recreational athletes Fernando Naclerio, Eneko Larumbe-Zabala, Robert Cooper, Alfonso Jimenez, and Mark Goss-Sampson

Abstract: Carbohydrate-protein–based multi-ingredient supplements have been proposed as an effective strategy for limiting the deleterious effects of exercise-induced muscle damage. This study compares the effects of a commercially available carbohydrate– protein supplement enriched with L-glutamine and L-carnitine-L-tartrate to carbohydrate alone or placebo on sprint performance, muscle damage markers, and recovery from intermittent exercise. On 3 occasions, 10 recreationally trained males ingested a multiingredient, a carbohydrate supplement, or a placebo before, during, and immediately after a 90-min intermittent repeated sprint test. Fifteen-metre sprint times, creatine kinase, myoglobin, and interleukin-6 were assessed before (pre), immediately after (post), 1 h after (1h), and 24 h after (24h) exercise. Total sprint time measured during the intermittent protocol was not different between conditions. Fifteen-metre sprint time was slower (p < 0.05) at post, 1h and 24h compared with pre without differences between conditions (p > 0.05). Creatine kinase at 24h was lower (p < 0.05) in the multi-ingredient (461.8 ± 271.8 U·L) compared with both carbohydrate and placebo (606 ± 314.5 U·L and 636 ± 344.6 U·L, respectively). Myoglobin increased (p < 0.05) in all 3 conditions at post and 1h compared with pre, showing lower values at 1h (p < 0.05) for the carbohydrate and a trend (p = 0.060) for multi-ingredient compared with the placebo condition (211.4 ± 127.2 ng·mL−1 and 239.4 ± 103.8 ng·mL−1 vs. 484.6 ± 200.0 ng·mL−1, respectively). Interleukin-6 increased at both post and 1h compared with pre (p < 0.05) with no differences between conditions. In conclusion, ingesting a multi-ingredient supplement before, during, and immediately after a 90-min intermittent sprint test resulted in no effects on performance and fatigue while the accumulation of some biomarkers of muscle damage could be attenuated. Key words: nutritional supplements, creatine kinase, myoglobin, interleukin-6, muscle fatigue. Résumé : La polysupplémentation comprenant des sucres et des protéines est proposée par des auteurs a` titre de stratégie efficace pour limiter les effets nuisibles des lésions musculaires suscitées par l’exercice physique. Afin d’en mesurer les effets sur la performance au sprint, les marqueurs de lésion musculaire et la récupération d’un exercice intermittent, cette étude compare l’ingestion d’un supplément de sucres et d’un placebo a` un supplément, disponible sur le marché, a` base de sucres et de protéines enrichi de L-glutamine et de L-tartrate de L-carnitine. En trois occasions, 10 hommes s’entraînant par loisir consomment un multisupplément, un supplément ou un placebo avant, pendant et immédiatement après un test de 90 min de sprint répété. Avant l’exercice et immédiatement après puis 1 h et 24 h plus tard, on évalue le temps de performance au sprint, la créatine kinase, la myoglobine et l’interleukine-6. D’une condition a` l’autre, on n’observe pas de différence de temps total de sprint dans le protocole intermittent. Le temps de performance au sprint de 15 m est plus long (p < 0,05) immédiatement après et 1 h et 24 h plus tard comparativement au temps avant, et ce, sans différence d’une condition a` l’autre (p > 0,05). Il y a moins de créatine kinase (p < 0,05) 24 h plus tard dans la condition de multisupplément (461,8 ± 271,8 U·L) comparativement a` la condition de sucre (606 ± 314,5 U·L) et du placebo (636 ± 344,6 U·L). La concentration de myoglobine augmente (p < 0,05) dans les trois conditions immédiatement et 1 h après comparativement aux valeurs initiales, mais présente de plus faibles valeurs 1 h après (p < 0,05) dans la condition de sucres et une tendance (p = 0,060) dans la condition de multisupplément comparativement a` la condition placebo (211,4 ± 127,2 ng·mL−1 et 239,4 ± 103,8 ng·mL−1 vs 484,6 ± 200,0 ng·mL−1, respectivement). L’interleukine-6 augmente immédiatement et 1 h après l’exercice comparativement aux valeurs initiales (p < 0,05), mais sans différence entre les conditions. En conclusion, la consommation d’un multisupplément avant, durant et immédiatement après un test de 90 min de sprint intermittent n’a pas d’effet sur la performance et la fatigue et suscite une légère diminution des quelques biomarqueurs de lésion musculaire. [Traduit par la Rédaction] Mots-clés : supplément nutritif, créatine kinase, myoglobine, interleukine-6, fatigue musculaire.

Introduction Exercise induced muscle damage (EIMD) is produced when an individual is exposed to a repeated bout of unaccustomed movement (Schoenfeld 2012). Damage can be variable and ranges from the isolated disruption of a few sarcomeres to tears in the sarcolemma basal

lamina and supportive connective tissue, all of which may alter the function of contractile elements and the cytoskeleton. Damage to the myofibres, in addition to being associated with the alteration of the excitation–contraction coupling system and sarcomere disruption, can also result in the release of intracellular proteins and influx

Received 3 December 2013. Accepted 24 April 2014. F. Naclerio, R. Cooper, and M. Goss-Sampson. Centre for Sport Science and Human Performance, School of Science, University of Greenwich, Medway Campus Central Avenue, Chatham Maritime, Kent ME4 4TB, UK. E. Larumbe-Zabala. Fundamentals of Motricity and Sports Training Department, School of Sports Sciences, European University of Madrid, Madrid, Spain. A. Jimenez. Fundamentals of Motricity and Sports Training Department, School of Sports Sciences, European University of Madrid, Madrid, Spain; Institute of Sport, Exercise and Active Living (ISEAL), Victoria University, Melbourne, Australia. Corresponding author: Fernando Naclerio (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 1151–1158 (2014) dx.doi.org/10.1139/apnm-2013-0556

Published at www.nrcresearchpress.com/apnm on 5 May 2014.

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of extracellular proteins leading to cell swelling, disturbance of the extracellular matrix and mediation of the inflammatory response associated with further functional muscle impairment (Friden and Lieber 2001; Schoenfeld 2012). Structural damage to the muscle cell is accompanied by the leakage of proteins such as creatine kinase (CK) and myoglobin (Mb) out of the cell and into the circulation (Peake et al. 2005). Thompson et al. (1999) observed significant increases of CK (379% from baseline) that peaked at 24 h after performing 90-min of the Loughborough Intermittent Shuttle Test (LIST) in 16 young males. Similarly, Twist and Eston (2005) reported significant rises of CK values coupled with slower 10-m sprint times observed 24 h after performing a battery of sprint tests in 10 young male college athletes. Elevated plasma Mb after 30 min and CK levels throughout the 72-h recovery period were also reported after performing a 90-min LIST and a friendly match in football players (Ascensao et al. 2008; Magalhaes et al. 2010). EIMD may also result in the release of several plasma cytokines into the circulation (Pedersen 2011). Elevated levels of skeletal muscle-derived interlukin-6 (IL-6) into the blood appears to be the major alteration in cytokines observed during exercise (Thiago Gomes et al. 2012). Furthermore, Leeder et al. (2013) reported a decrease in vertical jump performance in trained young males together with increased levels of plasma CK and IL-6 peaking at 24 h and immediately after performing the LIST. Some protective strategies, including nutritional interventions, have been proposed to attenuate the negative consequences of the EIMD (Howatson and van Someren 2008). In fact, the consumption of carbohydrate (CHO) alone and in combination with protein supplements has been shown to be effective for improving performance, attenuating fatigue, promoting of the recovery process, and reducing markers of muscular damage when ingested before (Kerksick et al. 2008; de Sousa et al. 2012), during (de Sousa et al. 2012), and after exercise (Cockburn et al. 2008, 2010; Costa et al. 2011). With regards to intermittent exercise involving frequent changes of direction, such as the acceleration and repeated sprints as seen during football or rugby, the consumption of natural supplements intended to attenuate performance decreases or enhance the recovery process have been well studied (Nieman and Bishop 2006). Alghannam (2011) observed positve effects of ingesting a CHO–protein supplement, providing 0.7 g·kg−1 and 0.3 g·kg−1, respectively, on intermittent running performance in 6 young male amateur football players. Other studies using intermittent exercise protocols, reported positive effects of L-glutamine or L-carnitine supplementation for attenuating oxidative stress and muscular damage, and hence favour performance and optimise recovery after training (Bassini-Cameron et al. 2008; Spiering et al. 2008). The ingestion of 100 mg·kg−1 of L-glutamine has been shown to reduce the accumulation of blood ammonia and possibly attenuate peripheral and central fatigue in young professional football players (Bassini-Cameron et al. 2008). On the other hand, 2 to 4 g·day−1 (Volek et al. 2002; Wall et al. 2011) of L-carnitine-L-tartrate have been shown to attenuate muscle damage and oxidative stress after performing a muscle damaging exercise protocol. To summarise, the ingestion of CHO alone or combined with protein or other nutrients such as L-carnitine or L-glutamine has shown the potential to positively influence performance and promote recovery from strenuous exercise. In recreational athletes, the main reasons for using protein enriched supplements are to increase muscle mass, improve exercise recovery, and to enhance performance (Erdman et al. 2007). However, even though scientific information is available, supplement users rely more on coaches, teammates, family, or friends to obtain advice on these products (Bianco et al. 2011). The final decision appears to be mainly based on the marketing claims, rather than on the available evidence-based research (McLellan et al. 2014). The use of CHO and protein-based multi-ingredient supplements has recently increased, particularly by those playing recreational team sports. The main reason for using these types of mixed supplements

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

rely on the belief that adding protein to CHOs will not only support performance but also speed recovery (Darvishi et al. 2013). The aim of this study, therefore, was to compare the sprint performance, recovery, and muscle-protective effects of a commercially available combined CHO and protein-based multi-ingredient supplement enriched with L-glutamine and L-carnitine-L-tartrate (multinutrient (MTN)) with a CHO only supplement and a low-calorie placebo (PL). These effects were monitored in recreationally trained athletes over a 24-h postexercise period. Based on previous studies it was hypothesised that the ingestion of a CHO, protein, and amino acid multi-ingredient supplement before, during, and after an acute bout of an intermittent, repeated sprint exercise would improve performance, attenuate fatigue, and reduce markers of muscle damage compared with the ingestion of CHO only or a low-calorie PL.

Material and methods Participants Ten healthy males that were recreationally active in team sports (age, 25 ± 3.8 years; height, 182 ± 6.9 cm; body mass, 79.5 ± 9.4 kg) volunteered to participate in the study, providing written informed consent in accordance with the Declaration of Helsinki. All participants were screened for any musculoskeletal injuries, metabolic conditions, or diseases; and use of medications, smoking, and nutritional supplements known to affect muscle damage or recovery process (e.g., creatine, whey protein, and amino acids) within 6 weeks prior to the start of the study. All experimental procedures were evaluated and approved by the university’s ethics committee. Experimental design This study utilised a double-blind, single-group repeated measures design, where 3 within-participant conditions, MTN, CHO, and PL, were considered. Once considered eligible for the study, each participant was required to attend the laboratory on 6 different occasions. On the first visit participants were assessed for body mass, height, and maximal aerobic speed (MAS). The next 2 visits were intended to familiarise participants with the 90-min intermittent repeated sprint test (IRST) protocol. The remaining visits required participants to perform the IRST under the 3 assessed conditions: MTN, CHO, and PL. To maintain a suitable balance between all the possible orders of treatments and minimise any confounding effects, the order of the treatments was randomised in a controlled manner. Thus, a third of the participants started with treatment 1, a third with treatment 2, and the remaining third with treatment 3. The same arrangement was used for the allocation of the second and third treatment sessions. Five to 7 days were allowed between each of the 3 testing conditions. Participants were asked to abstain from any unaccustomed or hard exercise during the 72 h before each of the 3 main testing sessions. Procedures Prior to the IRSTs assessment, participants were required to provide a diet diary for 3 consecutive days consisting of 2 week days and 1 weekend day. Diets were then analysed for CHO, protein, fat, and energy content using Dietplan6 software (Forestfield Software, UK). Participants were instructed to maintain their normal diet throughout the intervention. Pre-exercise standardized meal Two hours before arriving to the lab, participants were required to consume a standardised meal sourced from porridge oats and semi-skimmed milk that provided 1 g·kg−1 CHO and 0.15 g·kg−1 protein. Experimental protocols Determination of MAS A standard multistage 20-m shuttle run test, starting at 8.5 km·h−1 and increasing by 0.5 km·h−1 every minute, was used to predict MAS Published by NRC Research Press

Naclerio et al.

(Leger and Lambert 1982). The test was halted when a participant failed to arrive at the line before the audible beep on 2 consecutive occasions. Verbal encouragement was given and a minimum score of level 8 had to be achieved for a participant to be considered suitable for the study. Furthermore, MAS was estimated from the last completed stage and used to pace sections of the IRST.

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Fig. 1. Schematic overview of the study design. !, MTN, CHO, or PL ingestion; 15m, sprint test; BT, beep test; F, familiarisation sessions; IRST, intermittent repeated sprint test involving 4 blocks of 11 sets of 3 repetitions of 60 m at 60%; 80%, and 60% maximal aerobic speed plus 15-m sprint. End

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Start

IRST After the baseline line blood sample and prior to the IRST, a standardised 5-min warm-up involving different shuttle run speeds and dynamic stretching was completed. The IRST was a modified version of the LIST (Nicholas et al. 2000) of approximately 90 min in duration and divided into 4 blocks with 3 min of rest between each block. Each block consisted of 11 cycles of 3 repetitions of 20 m of self-paced walking, below 60% of MAS, followed by one 15-m sprint after which 3 repetitions of 20 m of running at 80% of MAS and 3 repetitions of 20 m of jogging at 60% of MAS were performed. Therefore, a total of 44 cycles were completed for each IRST, covering a total distance of 8580 m at varying velocities. Fatigue assessment and biomarker measures were determined before the IRST (pre), immediately after IRST (post), 1 h after IRST (1h), and 24 h after IRST (24h). 15-m sprint test The 15-m sprint test was selected to specifically examine fatigue induced by the IRST (Nicol et al. 2006). Each participant performed three 15-m sprints; each sprint time was measured using an infrared timing gate system (Brower Timing Systems). After walking back to the start of the sprint track, participants were required to rest for 30 s between sprints to allow for recovery, and the best of the 3 sprints was used for the analysis. The coefficients of variation for this test, calculated from reliability trials conducted in previous pilot studies, are between 0.5% and 1%. Blood sampling and analysis Vacutainer venous blood collection tubes were used to collect 10 mL of heparinised blood and 10 mL of whole blood from the cubital fossa. Nonheparinised blood samples were inverted 5 times and allowed to stand for 1 h prior to being centrifuged at 3500 r·min–1, at a temperature of 4 °C, for 10 min, after which the serum was aliquoted into Eppendorf tubes and frozen at –70 °C for later analysis. Heparinised samples were inverted 8 times in the vacutainer to ensure adequate mixing with the heparin. Thirtytwo microlitres of heparinised blood was pipetted out onto a test strip and analysed, for CK, using a colorimetric assay procedure (Reflotron Boehringer Mannheim, Germany) (Horder et al. 1991). The remaining heparinised blood was centrifuged at 3500 r·min–1, at a temperature of 4 °C, for 10 min, after which the plasma was aliquoted into Eppendorf tubes and frozen at –70 °C for later analysis. IL-6 (R&D Systems; HS600B, Abingdon, UK) and Mb (Abcam; ab108652, Cambridge, UK) were each assayed in duplicate using an ELISA in accordance with the assay kit instructions provided by the manufacturer. As reported in the manufacturer’s data sheets, the intra-assay and inter-assay coefficients of variation for CK were 3.0% and 3.5%, respectively. For Mb assays, they were 3.5%– 6.0% and 5.0%–10.0%, respectively. Coefficients of variation for IL-6 were 6% to 8% and 10% to 12%. Supplementation Immediately prior to the first, second, third, and fourth blocks of the IRST, participants ingested 500 mL of water mixed with MTN, CHO, or PL that were divided and administered into 4 equal doses. Each 500-mL dose of MTN contained 53 g of CHO (maltodextrin and dextrose), 14.5 g of whey protein, 1.2 g fat, 5 g of glutamine, 1.5 g of L-carnitine-L-tartrate, and provided 280 kcal, whilst a 500-mL dose of CHO contained 69.5 g of CHO (maltodextrin) only and provided 265 kcal. The PL was a low kcal beverage (20.97 kcal per serving) of the same volume, colour, and flavour as

1x500ml

BT F F

1

2

3

4

Post

1h

24 h

4 x 125ml (500ml total)

15m Blood

15m 15m Blood Blood

15m Blood

MTN and CHO. A second full dose was provided 20 min after the IRST. A total of 2 full doses of MTN, CHO, or PL were ingested in each condition. Therefore, the MTN supplement provided a total of 106 g CHO, 29 g protein, 2.4 g fat, 10 g glutamine, 3 g L-carnitineL-tartrate, and 560 kcal, whilst the CHO supplement provided a total of 139 g CHO and 530 kcal. Figure 1 shows the structure of study protocol. Statistical analysis Data are presented as means ± SD. Mauchly’s Test of Sphericity was used for testing the normality distribution of the data. Twoway repeated measures ANOVA (3 test conditions × 4 sprint blocks) was performed to analyse the total sprint time (sum of 44 sprints) performed at IRST. The differences between conditions and time points were assessed using 2-way repeated measures ANOVA (3 test conditions × 4 time points). Bonferroni-adjusted post hoc analysis was performed for pairwise comparisons. To measure standardised effect size, omega squared (␻2) was used. In absence of specific thresholds from the literature, reference values (small (␩2 = 0.01), medium (␩2 = 0.06), and large (␩2 = 0.14)) from Cohen (1988) were considered. Significance level was set at P < 0.05 for all tests.

Results Diets Participants verbally confirmed that they maintained their habitual diet throughout the trial period. The average daily consumption of CHO, protein, fat, and energy was 4.2 ± 0.18, 1.5 ± 0.20, 1.04 ± 0.15 g·kg−1, and mean 31.2 ± 1.6 kcal·kg−1, respectively. IRST, repeated sprint performance The sum of eleven 15-m sprint times obtained per each of the 4 blocks and for the 44 total sprints performed for entire IRST was considered as indicators of sprint performance. Table 1 shows the total times summarised at blocks 1, 2, 3, and 4, as well as for the total IRST for each of the 3 analysed conditions (MTN, CHO, and PL). No significant main effects were observed when comparing the sum of the total time for the entire IRST per condition (MTN, CHO, or PL) (F[2,18] = 0.12, p = 0.891, ␻2 ≈ 0.00). Significant effect was observed when considering the time performed per block (1, 2, 3, and 4) (F[3,27] = 3.03, p = 0.047, ␻2 ≈ 0.14). However, no significant interaction effects between conditions and time per block were determined (F[6,54] = 1.57, p = 0.174, ␻2 ≈ 0.01). Post hoc analysis did not reveal any significant difference. Published by NRC Research Press

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Table 1. Total 15-m sprint times (s) per block and all the entire intermittent repeated sprint test.

MTN CHO PL

All 4 blocks (s)

Block 1 (s)

Block 2 (s)

Block 3 (s)

Block 4 (s)

106.81 (7.19) 107.58 (7.59) 107.50 (8.41)

26.44 (1.70) 26.31 (1.44) 26.71 (1.88)

26.57 (1.74) 26.82 (1.99) 26.73 (1.97)

27.14 (2.42) 27.23 (2.12) 26.93 (2.04)

26.66 (1.62) 27.23 (2.28) 27.13 (2.70)

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Note: Values are means (±SD). CHO, carbohydrate; MTN, multi-ingredient; PL, placebo.

15-m sprint test Table 2 shows the 15-m sprints performed at the 4 different time points (pre, post, 1h, and 24h) for the 3 analysed conditions (MTN, CHO, and PL). Significant main effects were observed for time (pre, post, 1h, and 24h), (F[3,27] = 20.21, p < 0.001, ␻2 = 0.20), but not between conditions (MTN, CHO, PL) (F[2,18] = 0.10, p = 0.904, ␻2 ≈ 0.00). Post hoc analysis revealed significantly (p < 0.05) longer sprint times for all 3 conditions at either post, 1h, and 24h compared with pre but not between the sprint times determined at the 3 post-time points (post, 1h, and 24h). Additionally, no significant interaction effects between time and conditions were determined (F[6,54] = 1.28, p = 0.283, ␻2 ≈ 0.00). Muscle damage markers Table 3 shows the values determined for CK; Mb and IL-6 at the 4 different time points (pre, post, 1h, and 24h) for the 3 conditions (MTN, CHO, and PL). Before performing the IRST (pre), no significant differences between conditions (MTN, CHO, and PL) were observed for the 3 analysed markers: CK (F[2,16] = 4.23, p = 0.066); Mb (F[2,16] = 0.59, p = 0.485) and IL-6 (F[2,16] = 2.55, p = 0.146). However, some differences and interaction effects were determined for the measurement obtained at post, 1h, and 24h after performing the IRST. Significant effect of time points (pre, post, 1h, and 24h) (F[3,24] = 26.0, p < 0.001, ␻2 = 0.24) and interaction between conditions and times (F[6,48] = 2.79, p = 0.021, ␻2 = 0.02) were determined for CK. Post hoc analysis revealed significant (p < 0.05) higher values of CK at 24h compared with all other time points for CHO and PL conditions but not for the MTN condition (Fig. 2). Mb analysis showed significant differences between conditions (MTN, CHO, and PL) (F[2,16] = 5.17, p = 0.019, ␻2 = 0.04), time (pre, post, 1h, and 24h) (F[3,24] = 49.65, p < 0.001, ␻2 = 0.49), and interactions effects between conditions and time (F[6,48] = 5.79, p < 0.001, ␻2 = 0.08). Post hoc analysis revealed significantly higher Mb values for all 3 conditions at post (MTN, p = 0.024; CHO, p = 0.013; and PL, p = 0.028) and 1h (MTN, p = 0.010; CHO, p = 0.001; and PL, p = 0.003) compared with pre. However, only PL showed significant higher values at 1h compared with post (p = 0.013). At 24h Mb decreased for all 3 conditions, reaching significantly lower values than those measured at 1h (MTN, p = 0.006; CHO, p = 0.001; PL, p < 0.001). However, MTN (p = 0.015) and CHO (p = 0.017), but not PL, showed significantly lower values at 24h compared with those measured at post (Fig. 2). In addition, significantly lower Mb values were determined at 1h for CHO compared with PL (p = 0.019) and a trend when the values measured for MTN were compared with those determined at PL (p = 0.060). The same trends were found for the 24h values when the Mb produced after MTN (p = 0.065) and CHO (p = 0.057) were compared with those measured after PL condition (Fig. 3). IL-6 analysis showed a significant difference for time (pre, post, 1h, and 24h) (F[3,24] = 40.31, p < 0.001, ␻2 = 0.30). However, no differences were found for condition effect (F[2,16] = 1.81, p = 0.196, ␻2 ≈ 0.00) or its interaction with the time points (F[6,48] = 1.78, p = 0.124, ␻2 ≈ 0.00). Post hoc analysis revealed higher IL-6 values for all 3 conditions at both post (MTN, p = 0.001; CHO, p = 0.001; PL, p = 0.0130) and 1h (MTN, p < 0.001; CHO, p = 0.001; PL, p = 0.005) compared with pre. In addition, at 24h the IL-6 values seemed to approach baseline values, being significantly lower than those

Table 2. Fifteen-metre sprint times (s) measured at before (pre), immediately after (post), and 1 h (1h) and 24 h (24h) after intermittent repeated sprint test. 15 m (s)

MTN CHO PL

Pre

Post

1h

24h

2.44 (0.2) 2.40 (0.2) 2.45 (0.15)

2.65 (0.2) 2.63 (0.2) 2.62 (0.2)

2.63 (0.2) 2.67 (0.2) 2.59 (0.1)

2.58 (0.2) 2.61 (0.1) 2.59 (0.15)

Note: Values are means (±SD). CHO, carbohydrate; MTN, multi-ingredient; PL, placebo.

measured at both post (MTN, p = 0.001; CHO, p = 0.001; PL, p = 0.010) and 1h (MTN, p = 0.000; CHO, p = 0.000; PL, p = 0.003) after performing the IRST but not from those determined at pre (Fig. 4). No significant differences were observed when the IL-6 values measured at 24h were compared with those obtained at pre for the 3 analysed conditions.

Discussion The findings of this study demonstrate that there was no effect of ingesting an MTN or CHO supplement before, during, and immediately after exercise on repeated sprint ability, 15-m sprint test, or pattern of serum IL-6 responses measured at post, 1h, and 24h after performing a 90-min IRST in recreationally trained males. However, the ingestion of an MTN supplement appears to be effective in blunting the increase in CK observed 24 h after the IRST. In addition, changes in Mb were observed following consumption of both MTN and CHO compared with PL. These results are in contrast to a number of studies that have suggested positive effects of CHO-protein mixtures, compared with the ingestion of CHO alone or a low-calorie PL, for improving intermittent endurance performance (Alghannam 2011) and the attenuation of neuromuscular fatigue (Cockburn et al. 2010, 2012). Although the total amount of protein in the MTN condition was greater than that recommended as effective for attenuating fatigue (Cockburn et al. 2012) or to maximally stimulate muscle and albumin protein synthesis after performing an acute bout of lower body resistance exercises (Moore et al. 2009). The benefit induced by the ingestion of CHO-protein supplements during prolonged exercises is based on the rationale that the added protein would promote reliance on exogenous carbohydrate oxidation and reduce glycogen depletion, thereby improving performance times and hasten the recovery of muscle glycogen stores (McLellan et al. 2014). This positive effect would be also supported by the increase in muscle protein synthesis and (or) blunting protein degradation (Cockburn et al. 2010). As stated by McLellan et al. (2013), adding protein to CHO improves performance during an acute bout of endurance exercise when CHO is provided at less than optimal rate of 60 g·h−1. However, when CHO supplementation is delivered at or above the optimal rate no further ergogenic effect has been reported by the ingestion of added proteins. In the present study participants were ingesting 53 g and 46 g of CHO during 90-min exercises at the CHO and MTN conditions, respectively. Thus, in both cases the rate of CHO delivered during exercise was set below the optimal (46 g·h−1 and 35.5 g·h−1, respectively). This would probably have influenced the lack of ergogenic effect on performance observed Published by NRC Research Press

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Table 3. Muscle damage markers determined at before (pre), immediately after (post), and 1 h (1h) and 24 h (24h) after intermittent repeated sprint test. Mb (ng·mL−1)

CK (U·L) Pre

Post

1h

24h

Pre

IL-6 (pg·mL−1)

Post

1h

24h

Pre

Post

1h

24h

MTN 230.4 (60.0) 349.9 (140.5) 401.2 (196.7) 461.8* (271.8) 7.2 (10.9) 181.9 (126.6) 211.4 (127.2) 4.7 (8.5) 1.2 (2.6) 4.7 (2.5) 4.0 (2.3) 1.3 (2.4) CHO 151.5 (61.4) 269.6 (70.0) 313.0 (97.7) 606.6 (314.5) 3.5 (4.8) 218.7 (145.1) 239.4 (103.8) 13.2 (18.2) 1.0 (2.1) 4.1 (2.4) 3.9 (2.4) 1.1 (2.2) PL 147.4 (68.4) 307.6 (134.1) 356.3 (172.7) 636.3 (344.6) 32.7 (51.9) 162.8 (136.9) 484.6† (200.0) 88.1 (86.2) 1.1 (2.7) 4.8 (3.0) 4.7 (2.8) 1.3 (2.7)

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Note: Values are means (±SD). CHO, carbohydrate; CK, creatine kinase; Mb, myoglobin; MTN, multi-ingredient; PL, placebo. *p < 0.05 compared with CHO and PL. †p < 0.05 compared with CHO.

Fig. 2. Serum creatine kinase (CK) levels determined at all time points for the 3 tested conditions. *, p < 0.05 from 24 h after exercise (24 hr) compared with before (pre), immediately after (post), and 1 h after (1 hr) exercise for carbohydrate (CHO) and placebo (PL) conditions. MTN, muti-ingredient.

*

Fig. 3. Serum myoglobin (Mb) values determined at all times points for the 3 conditions. *, p < 0.05 compared with before exercise (pre) for the 3 conditions. ␰, p < 0.05 from 1 h after exercise (1hr) compared with immediately after exercise (post) only for the placebo (PL) condition. ⍀, p < 0.05 compared with all other time points for the 3 conditions. ␦, p < 0.05 compared with post for muti-ingredient (MTN) and carbohydrate (CHO) conditions.

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* Ω δ at both conditions. Even when in the MTN condition, the addition of protein to a suboptimal amount of CHO would be expected to improve performance (McLellan et al. 2014) as the total energy provided in both conditions was almost similar and probably would have been not enough to elicit performance improvement. These results support the notion that to attenuate performance loss during intermittent endurance exercise, the most relevant factor is the energetic content of the supplement rather than mixing protein and CHOs (McLellan et al. 2014). Regardless of the effect on performance, the benefits of adding protein to CHO supplements appears to be based on the efficacy to increase protein synthesis, attenuate protein degradation, and protect against muscle membrane disruption (Cockburn et al. 2010). Nevertheless, these positive effects have been observed mainly after an acute bout of resistance exercises (Tang et al. 2007; Cockburn et al. 2008, 2010, 2012; Moore et al. 2009) and not when

performing an intermittent high-intensity running exercise as performed by our participants. Witard et al (2014) have recently demonstrated that muscle protein synthesis is not further stimulated with intakes of whey protein above 20 g after performing 8 sets of 10 repetitions at 80% 1-repetition maximum of an unilateral bout of 2 lower body exercises. This amount of protein would set an optimal value for achieving the maximal response beyond which a marked stimulation of whole-body amino acid oxidation and ureagenesis with no further increases in muscle protein synthesis would occur. However, these studies have administered protein as 1 bolus within ⬃10 min after the workout (Tang et al. 2007; Moore et al. 2009; Witard et al. 2014) and not spread out along the exercise as occurred in our study. Probably, even when the total amount of protein provided in the MTN condition (29 g) was greater than the optimal value needed to maximally stimulate muscle protein synthesis, the consumption pattern used in Published by NRC Research Press

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Fig. 4. Comparison between the serum myoglobin (Mb) levels determined for the 3 tested conditions at 1 h (1hr) and 24 h (24hr) after performing the intermittent repeated sprint test. *, p < 0.05 compared with carbohydrate (CHO). ␦, p = 0.060 compared with muti-ingredient (MTN). ⌽, Trend to CHO (p = 0.057) and to MTN (p = 0.065). PL, placebo.

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*δ

Φ

our study, which is more similar to what team athletes can do, would not be effective to elicit this response in recreationally trained males when performing a 90-min IRST (West et al. 2011). With regard to the muscle damage markers, total CK levels measured at 24 h were significantly reduced in MTN compared with CHO and PL whereas both MTN and CHO showed lower Mb values than PL at 1 h with no difference between them. However, total CK is a highly variable, indirect, and nonspecific marker of exercise-induced muscular damage (Brancaccio et al. 2010) and Mb should also be used with caution (Sorichter et al. 2001). In fact, the analysis used for the assay of both markers does not distinguish between skeletal or cardiac muscle isoforms. In addition, substantial variation between participants (Table 3) together with the lack of homogeneity in the observed response for the 3 tested conditions makes it difficult to differentiate any muscular protective effects because of the ingestion of MTN or CHO alone and hence challenges their use as meaningful markers of exercise induced muscular damage. However, the significant increase of IL-6 observed immediately post and 1h after exercise but not at 24h (when Il-6 levels approach baseline), without differences between conditions, impede the elucidation of any further protective effects because of the ingestion of MTN or CHO compared with PL. Indeed, it has been suggested that the increase in IL-6 following exercise is not primarily related to muscle damage but with the exercise volume (Phillips et al. 2010) and intensity (Ostrowski et al. 2000). During exercise, active muscles secrete IL-6 to influence and regulate substrate metabolism. IL-6 stimulates lipolysis and hepatic glycogenolysis as exercise progresses and muscular glycogen becomes progressively depleted (Pedersen and Febbraio 2008). Hence we would expect a blunted IL-6 response after the MTN or CHO conditions; however, no effects were observed compared with PL. After exercise, circulating IL-6 should decrease approaching the baseline levels within a few hours. However, some degree of muscular damage can be inferred when a sustained elevation of circulating Il-6 is observed from 6 h to several days after exercise (Lancaster 2006). The result of the present study are also similar to those reported by Roberts et al. (2011) who found no effects of ingesting a solution, providing 1.2 g·kg−1·h−1 of CHO alone or combined with 0.4 g·kg−1·h−1 of high-quality protein, before, during, and after a rugby-specific shuttle running protocol to attenuate muscular function and serum Mb accumulation measured immediately after exercise. In addition, similar to the present study, total CK peaked 24 h after exercise with no differences between the 3 analyzed conditions. More recently, Cockburn et al. (2013) reported that consuming 500 mL of semi-skimmed milk, providing 13.6 g of casein, 3.5 g of whey proteins, 24.5 g of CHO, and 8.5 g of fats, after an acute isoki-

netic concentric–eccentric hamstring muscle damage protocol, is likely to be beneficial in limiting the loss of performance during an agility task, 15-m sprint, and a 90-min repeated sprint test but not for attenuating the decrease of reactive strength and the increase of both serum Mb and CK measured over a 72-h period. In the present study, the consumption of a MTN formula, providing a total amount of 29 g of high-quality protein mixed with 106 g of CHO and enriched with 10 g of L-glutamine and 3 g of L-carnitine-L-tartrate, did not appear to be effective in reducing muscle damage when performing the 90-min IRST. In addition to the mechanical stress placed on the lower limbs because of the repetitive sprints (Gleeson et al. 1998), the level of glycogen depletion would seriously impact on performance and recovery processes (McLellan et al. 2014). Differences in the exercise protocol used in the present study, which involves repeated running maximal acceleration and decelerations over a 90-min test, would have increased the need for a higher amino acids supply to determine a more noticeable effect on muscular function and markers of muscle damage. However, this is purely speculative as there is no conclusive evidence regarding the dose–response relationship between the effects of ingesting CHO-protein based multi-ingredient supplements and performance or muscle damage markers. The addition of L-glutamine and L-carnitine-L-tartrate does not appear to have a significant effect on sprint performance during the IRST or the recovery period compared with CHO alone or PL. There was also no effect on Mb levels since these were similar in both the MTN and CHO conditions. The attenuation of CK at 24h could be considered but it is not possible to identify if this effect is due to the administration of protein, the added L-glutamine and L-carnitine, or a combination of different nutrients. Even though published results with respect to the effectiveness of the L-glutamine supplementation on performance are inconclusive (Kreider et al. 2010), some effects to prevent excessive muscle damage and neutrophil function suppression have been observed after several days of supplementation (Sasaki et al. 2013) but not after an acute intake as administered in the present study. With regard to L-carnitine, previous research demonstrated that 1 g·day−1 (Spiering et al. 2007) or 2 g·day−1 (Spiering et al. 2007, 2008) of L-carnitine-L-tartrate administered for 3 weeks results in less metabolic stress and concentration of both Mb (Spiering et al. 2007) and CK (Volek et al. 2002) measured after performing an acute bout of resistance exercise in young males. It is important to highlight that the above reported benefits obtained from the oral L-carnitine ingestion were observed after 21 days of regular administration and not after an acute intake. In addition, as plasma L-carnitine peaks after 3 to 6 h of ingestion (Rebouche 2004), to promote its benefits multiple daily doses where the last single intake should be administered around 3 h before exercise has been Published by NRC Research Press

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Naclerio et al.

recommended (Rebouche 2004). Thus, it could be possible that the protocol used in our study, where participants were supplemented during and after exercise, would not favour the positive effects of L-carnitine to acutely attenuate muscle damage as observed by others (Volek et al. 2002; Spiering et al. 2007). Although speculative, even when MTN or CHO did not improve repeated sprint ability during IRST or reduce the increase of 15-m sprint time measured over a 24-h period, as suggested by previous investigations (Volek et al. 2002; Spiering et al. 2007), the lower concentration of CK and Mb observed after exercise may be positively related to a faster recovery process in recreationally trained athletes. A limitation of this study was that only 3 time points postexercise were analyzed (immediately postexercise and 1 and 24 h after exercise) and therefore no further information about a longer recovery period (>24h) was obtained. Additionally, other markers were not analyzed, such as plasma aminoacidemia, glycaemia, serum glutamine, and carnitine concentrations. The current study measured IL-6 released into the blood and not locally and given the paracrine and autocrine function of IL-6, future studies could use microdialysis techniques or muscle biopsies to better analyse this parameter. Finally, even when acute studies can give a clear picture about the acute response resulting from different training and feeding protocols, to have more useful information about the potential benefit of natural supplements more research conducted over a longer period of time (weeks or months) should be designed. In conclusion, the ingestion of an MTN (53 g of CHO, 14.5 g of whey protein, 1.5 g of L-carnitine, and 5 g of L-glutamine) or CHO (69.5 g maltodextrin), dosed during and immediately after a 90-min intermittent repeated sprint exercise bout was not effective to increase performance, attenuate fatigue, or alter the IL-6 responses measured at post and 1 and 24 h after exercise in recreationally trained males. However, supplementing with MTN would be effective to blunt the increase in CK observed 24 h after the IRST with respect to CHO and PL. Finally, both MTN and CHO appear to have a similar positive effect in blunting the Mb increase observed 1 h after performing an IRST.

Acknowledgement The authors thank all participants, Kelly Cooper, Mark Chapman, Sergio Garcia, and Tobia Zanotto, Dan Baddeley, Marios Demetriou, and Mark Ward for their help and support. Maxinutrition and the University of Greenwich are providing joint funding to one of the author’s PhD project; however, this does not affect this original research article and its content.

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