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Luc J.C. van Loon

Protein, protein hydrolysates, and amino acids have become popular ingredients in sports nutrition. The use of protein, protein hydrolysates, and amino acid mixtures has multiple applications when aiming to improve post exercise recovery. After exhaustive endurance-type exercise, muscle glycogen repletion is the most important factor determining the time needed to recover. Coingestion of relatively small amounts of protein and/or amino acids with carbohydrate can be used to augment postprandial insulin secretion and accelerate muscle glycogen synthesis rates. Furthermore, it has been well established that ingesting protein, protein hydrolysates, and amino acid can stimulate protein synthesis and inhibit protein breakdown and, as such, improve net muscle protein balance after resistance- or endurance-type exercise. The latter has been suggested to lead to a more effective adaptive response to each successive exercise bout. To augment net muscle protein accretion, athletes involved in resistance-type exercise generally ingest both protein and carbohydrate during post exercise recovery. However, carbohydrate ingestion after resistance-type exercise does not seem to be warranted to further stimulate muscle protein synthesis or improve whole-body protein balance when ample protein has already been ingested. Because resistance-type exercise is also associated with a substantial reduction in muscle glycogen content, it would be preferred to coingest some carbohydrate when aiming to accelerate glycogen repletion. More research is warranted to assess the impact of ingesting different proteins, protein hydrolysates, and/or amino acids on muscle protein accretion after exercise.

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Luc J.C. van Loon, Arie K. Kies and Wim H.M. Saris

With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their effcacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28–29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

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Milou Beelen, Louise M. Burke, Martin J. Gibala and Luc J.C. van Loon

During postexercise recovery, optimal nutritional intake is important to replenish endogenous substrate stores and to facilitate muscle-damage repair and reconditioning. After exhaustive endurance-type exercise, muscle glycogen repletion forms the most important factor determining the time needed to recover. Postexercise carbohydrate (CHO) ingestion has been well established as the most important determinant of muscle glycogen synthesis. Coingestion of protein and/or amino acids does not seem to further increase muscle glycogensynthesis rates when CHO intake exceeds 1.2 g · kg−1 · hr−1. However, from a practical point of view it is not always feasible to ingest such large amounts of CHO. The combined ingestion of a small amount of protein (0.2–0.4 g · (0.2−0.4 g · kg−1 · hr−1) with less CHO (0.8 g · kg−1 · hr−1) stimulates endogenous insulin release and results in similar muscle glycogen-repletion rates as the ingestion of 1.2 g · kg−1 · hr−1 CHO. Furthermore, postexercise protein and/or amino acid administration is warranted to stimulate muscle protein synthesis, inhibit protein breakdown, and allow net muscle protein accretion. The consumption of ~20 g intact protein, or an equivalent of ~9 g essential amino acids, has been reported to maximize muscle protein-synthesis rates during the first hours of postexercise recovery. Ingestion of such small amounts of dietary protein 5 or 6 times daily might support maximal muscle protein-synthesis rates throughout the day. Consuming CHO and protein during the early phases of recovery has been shown to positively affect subsequent exercise performance and could be of specific benefit for athletes involved in multiple training or competition sessions on the same or consecutive days.

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Hermann Zbinden-Foncea, Luc J. C. van Loon, Jean-Marc Raymackers, Marc Francaux and Louise Deldicque

Mitogen-activated protein kinase (MAPK) pathways are activated in skeletal muscle during endurance exercise, but the upstream molecular events are incompletely resolved. As an increase in plasma nonesterified fatty acids (NEFA) is a common feature of long-lasting exercise, the authors tested the hypothesis that NEFA contribute to the activation of MAPK during endurance exercise. Acipimox was used before and during endurance exercise to prevent the elevation of plasma NEFA levels in healthy subjects and patients with diabetes. In 2 separate studies, healthy subjects cycled for 2 hr and patients with diabetes for 1 hr at 50% Wmax. In control conditions, plasma NEFA concentrations increased from 0.35 to 0.90 mM during exercise in healthy subjects and from 0.55 to 0.70 mM in patients with diabetes (p < .05). Phosphorylation states of extracellularly regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun NH2-terminal kinases (JNK) were significantly increased after exercise in the vastus lateralis in both groups. Acipimox blocked the increase in plasma NEFA concentrations and almost completely repressed any rise in ERK1/2 and p38 but not in JNK. In conclusion, the data support a role for plasma NEFA in the activation of p38 and ERK1/2 in skeletal-muscle tissue of healthy and diabetic subjects during endurance exercise. Further investigation will be required to determine the molecular link between NEFA and MAPK activation during exercise in human skeletal muscle.

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Naomi M. Cermak, Martin J. Gibala and Luc J.C. van Loon

Six days of dietary nitrate supplementation in the form of beetroot juice (~0.5 L/d) has been reported to reduce pulmonary oxygen uptake (VO2) during submaximal exercise and increase tolerance of high-intensity work rates, suggesting that nitrate can be a potent ergogenic aid. Limited data are available regarding the effect of nitrate ingestion on athletic performance, and no study has investigated the potential ergogenic effects of a small-volume, concentrated dose of beetroot juice. The authors tested the hypothesis that 6 d of nitrate ingestion would improve time-trial performance in trained cyclists. Using a double-blind, repeated-measures crossover design, 12 male cyclists (31 ± 3 yr, VO2peak = 58 ± 2 ml · kg−1 · min−1, maximal power [Wmax] = 342 ± 10 W) ingested 140 ml/d of concentrated beetroot (~8 mmol/d nitrate) juice (BEET) or a placebo (nitrate-depleted beetroot juice; PLAC) for 6 d, separated by a 14-d washout. After supplementation on Day 6, subjects performed 60 min of submaximal cycling (2 × 30 min at 45% and 65% Wmax, respectively), followed by a 10-km time trial. Time-trial performance (953 ± 18 vs. 965 ± 18 s, p < .005) and power output (294 ± 12 vs. 288 ± 12 W, p < .05) improved after BEET compared with PLAC supplementation. Submaximal VO2 was lower after BEET (45% Wmax = 1.92 ± 0.06 vs. 2.02 ± 0.09 L/min, 65% Wmax 2.94 ± 0.12 vs. 3.11 ± 0.12 L/min) than with PLAC (main effect, p < .05). Wholebody fuel selection and plasma lactate, glucose, and insulin concentrations did not differ between treatments. Six days of nitrate supplementation reduced VO2 during submaximal exercise and improved time-trial performance in trained cyclists.

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Evelien Backx, Cindy van der Avoort, Michael Tieland, Kamiel Maase, Arie Kies, Luc van Loon, Lisette de Groot and Marco Mensink

Studies monitoring vitamin D status in athletes are seldom conducted for a period of 12 months or longer, thereby lacking insight into seasonal fluctuations. The objective of the current study was to identify seasonal changes in total 25-hydroxyvitamin D (25(OH)D) concentration throughout the year. Fifty-two, mainly Caucasian athletes with a sufficient 25(OH)D concentration (>75 nmol/L) in June were included in this study. Serum 25(OH)D concentration was measured every three months (June, September, December, March, June). In addition, vitamin D intake and sun exposure were assessed by questionnaires at the same time points. Highest total 25(OH)D concentrations were found at the end of summer (113 ± 26 nmol/L), whereas lowest concentrations were observed at the end of winter (78 ± 30 nmol/L). Although all athletes had a sufficient 25(OH)D concentration at the start of the study, nearly 20% of the athletes were deficient (<50 nmol/L) in late winter.

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Kristin L. Jonvik, Jean Nyakayiru, Jan-Willem van Dijk, Floris C. Wardenaar, Luc J.C. van Loon and Lex B. Verdijk

Although beetroot juice, as a nitrate carrier, is a popular ergogenic supplement among athletes, nitrate is consumed through the regular diet as well. We aimed to assess the habitual dietary nitrate intake and identify the main contributing food sources in a large group of highly trained athletes. Dutch highly trained athletes (226 women and 327 men) completed 2–4 web-based 24-hr dietary recalls and questionnaires within a 2- to 4-week period. The nitrate content of food products and food groups was determined systematically based on values found in regulatory reports and scientific literature. These were then used to calculate each athlete’s dietary nitrate intake from the web-based recalls. The median[IQR] habitual nitrate intake was 106[75–170] mg/d (range 19–525 mg/d). Nitrate intake correlated with energy intake (ρ = 0.28, p < .001), and strongly correlated with vegetable intake (ρ = 0.78, p < .001). In accordance, most of the dietary nitrate was consumed through vegetables, potatoes and fruit, accounting for 74% of total nitrate intake, with lettuce and spinach contributing most. When corrected for energy intake, nitrate intake was substantially higher in female vs male athletes (12.8[9.2–20.0] vs 9.4[6.2–13.8] mg/MJ; p < .001). This difference was attributed to the higher vegetable intake in female vs male athletes (150[88–236] vs 114[61–183] g/d; p < .001). In conclusion, median daily intake of dietary nitrate in highly trained athletes was 106 mg, with large interindividual variation. Dietary nitrate intake was strongly associated with the intake of vegetables. Increasing the intake of nitrate-rich vegetables in the diet might serve as an alternative strategy for nitrate supplementation.

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Kristin L. Jonvik, Jan-Willem van Dijk, Joan M.G. Senden, Luc J.C. van Loon and Lex B. Verdijk

Nitrate-rich beetroot juice is thought to have ergogenic effects, particularly in conditions where oxygen availability is limited. Whether these effects also apply to elite athletes is currently unknown. The aim of this study was to assess the effects of beetroot juice supplementation on dynamic apnea and intermittent sprint performance in elite female water polo players. In a double-blinded, randomized, crossover manner, the Dutch National female water polo team (N = 14) was subjected to two 6-day supplementation periods (1 and 2), with either 140 ml/day of nitrate-rich (BR; ∼800 mg/day nitrate) or nitrate-depleted (PLA) beetroot juice. Following blood sampling on Day 6, the athletes performed a maximal-distance front crawl swimming test without breathing (dynamic apnea test). In addition, intermittent sprint performance was assessed by performing 16 swim sprints of 15 m, in a 4 × 4 block with 30-s recovery between blocks (intermittent test). Distance covered during the dynamic apnea test did not differ between BR (49.5 ± 7.8 m) and PLA (46.9 ± 9.1 m, p = .178). However, when correcting for test order, the distance covered was significantly larger in BR versus PLA when BR was ingested in Period 2 (50.1 ± 8.5 vs. 42.8 ± 5.7 m, p = .002), whereas no difference was observed when BR was ingested in Period 1 (48.8 ± 7.4 vs. 52.3 ± 10.4 m, p = .10). The time to complete the intermittent test was not different between BR and PLA (316.0 ± 7.9 vs. 316.3 ± 6.9 s, p = .73). In conclusion, beetroot juice supplementation does not improve intermittent performance in elite female water polo players, but there may be a potential for ergogenic effects during dynamic apnea.

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Jean M. Nyakayiru, Kristin L. Jonvik, Philippe J.M. Pinckaers, Joan Senden, Luc J.C. van Loon and Lex B. Verdijk

While the majority of studies reporting ergogenic effects of dietary nitrate have used a multiday supplementation protocol, some studies suggest that a single dose of dietary nitrate before exercise can also improve subsequent performance. We aimed to compare the impact of acute and 6-day sodium nitrate supplementation on oxygen uptake (V̇O2) and time-trial performance in trained cyclists. Using a randomized, double-blind, cross-over design, 17 male cyclists (25 ± 4 y, V̇O2peak 65 ± 4 ml·kg-1·min-1, Wmax 411 ± 35 W) were subjected to 3 different trials; 5 days placebo and 1 day sodium nitrate supplementation (1-DAY); 6 days sodium nitrate supplementation (6-DAY); 6 days placebo supplementation (PLA). Nitrate was administered as 1097 mg sodium nitrate providing 800 mg (~12.9 mmol) nitrate per day. Three hours after ingestion of the last supplemental bolus, indirect calorimetry was performed while subjects performed 30 min of exercise at 45% Wmax and 30 min at 65% Wmax on a cycle ergometer, followed by a 10 km time-trial. Immediately before exercise, plasma [nitrate] and [nitrite] increased to a similar extent during the 6-DAY and 1-DAY trial, but not with PLA (plasma nitrite: 501 ± 205, 553 ± 278, and 239 ± 74 nM, respectively; p < .001). No differences were observed between interventions in V̇O2 during submaximal exercise, or in time to complete the time-trial (6-DAY: 1004 ± 61, 1-DAY: 1022 ± 72, PLA: 1017 ± 71 s; p = .28). We conclude that both acute and 6-days of sodium nitrate supplementation do not alter V̇O2 during submaximal exercise or improve time-trial performance in highly trained cyclists, despite increasing plasma [nitrate] and [nitrite].

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James A. Betts, Milou Beelen, Keith A. Stokes, Wim H.M. Saris and Luc J.C. van Loon

Nocturnal endocrine responses to exercise performed in the evening and the potential role of nutrition are poorly understood. To gain novel insight, 10 healthy men ingested carbohydrate with (C+P) and without (C) protein in a randomized order and double-blind manner during 2 hr of interval cycling followed by resistancetype exercise and into early postexercise recovery. Blood samples were obtained hourly throughout 9 hr of postexercise overnight recovery for analysis of key hormones. Muscle samples were taken from the vastus lateralis before and after exercise and then again the next morning (7 a.m.) to calculate mixed-muscle protein fractional synthetic rate (FSR). Overnight plasma hormone concentrations were converted into overall responses (expressed as area under the concentration curve) and did not differ between treatments for either growth hormone (1,464 ± 257 vs. 1,432 ± 164 pg/ml · 540 min) or total testosterone (18.3 ± 1.2 vs. 17.9 ± 1.2 nmol/L · 540 min, C and C+P, respectively). In contrast, the overnight cortisol response was higher with C+P (102 ± 11 nmol/L · 540 min) than with C (81 ± 8 nmol/L · 540 min; p = .02). Mixed-muscle FSR did not differ between C and C+P during overnight recovery (0.062% ± 0.006% and 0.062% ± 0.009%/hr, respectively) and correlated significantly with the plasma total testosterone response (r = .7, p < .01). No correlations with FSR were apparent for the response of growth hormone (r = –.2, p = .4), cortisol (r = .1, p = .6), or the ratio of testosterone to cortisol (r = .2, p = .5). In conclusion, protein ingestion during and shortly after exercise does not modulate the endocrine response or muscle protein synthesis during overnight recovery.