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The Effect of Beetroot Juice Supplementation on Dynamic Apnea and Intermittent Sprint Performance in Elite Female Water Polo Players

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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No Improvement in Endurance Performance after a Single Dose of Beetroot Juice

Naomi M. Cermak, Peter Res, Rudi Stinkens, Jon O. Lundberg, Martin J. Gibala, and Luc J.C. van Loon


Dietary nitrate supplementation has received much attention in the literature due to its proposed ergogenic properties. Recently, the ingestion of a single bolus of nitrate-rich beetroot juice (500 ml, ~6.2 mmol NO3 ) was reported to improve subsequent time-trial performance. However, this large volume of ingested beetroot juice does not represent a realistic dietary strategy for athletes to follow in a practical, performancebased setting. Therefore, we investigated the impact of ingesting a single bolus of concentrated nitrate-rich beetroot juice (140 ml, ~8.7 mmol NO3 ) on subsequent 1-hr time-trial performance in well-trained cyclists.


Using a double-blind, repeated-measures crossover design (1-wk washout period), 20 trained male cyclists (26 ± 1 yr, VO2peak 60 ± 1 ml · kg−1 · min−1, Wmax 398 ± 7.7 W) ingested 140 ml of concentrated beetroot juice (8.7 mmol NO3 ; BEET) or a placebo (nitrate-depleted beetroot juice; PLAC) with breakfast 2.5 hr before an ~1-hr cycling time trial (1,073 ± 21 kJ). Resting blood samples were collected every 30 min after BEET or PLAC ingestion and immediately after the time trial.


Plasma nitrite concentration was higher in BEET than PLAC before the onset of the time trial (532 ± 32 vs. 271 ± 13 nM, respectively; p < .001), but subsequent time-trial performance (65.5 ± 1.1 vs. 65 ± 1.1 s), power output (275 ± 7 vs. 278 ± 7 W), and heart rate (170 ± 2 vs. 170 ± 2 beats/min) did not differ between BEET and PLAC treatments (all p > .05).


Ingestion of a single bolus of concentrated (140 ml) beetroot juice (8.7 mmol NO3 ) does not improve subsequent 1-hr time-trial performance in well-trained cyclists.

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A Sucrose Mouth Rinse Does Not Improve 1-hr Cycle Time Trial Performance When Performed in the Fasted or Fed State

Jorn Trommelen, Milou Beelen, Marjan Mullers, Martin J. Gibala, Luc J.C. van Loon, and Naomi M. Cermak

Carbohydrate mouth rinsing during exercise has been suggested to enhance performance of short (45–60 min) bouts of high-intensity (>75% VO2peak) exercise. Recent studies indicate that this performance enhancing effect may be dependent on the prandial state of the athlete. The purpose of this study was to define the impact of a carbohydrate mouth rinse on ~1-hr time trial performance in both the fasted and fed states. Using a double-blind, crossover design, 14 trained male cyclists (27 ± 6 years; 5.0 ± 0.5 W·kg−1) were selected to perform 4 time trials of ~1 hr (1,032 ± 127 kJ) on a cycle ergometer while rinsing their mouths with a 6.4% sucrose solution (SUC) or a noncaloric sweetened placebo (PLA) for 5 s at the start and at every 12.5% of their set amount of work completed. Two trials were performed in an overnight fasted state and two trials were performed 2 h after consuming a standardized breakfast. Performance time did not differ between any of the trials (fasted-PLA: 68.6 ± 7.2; fasted-SUC: 69.6 ± 7.5; fed-PLA: 67.6 ± 6.6; and fed-SUC: 69.0 ± 6.3 min; Prandial State × Mouth Rinse Solution p = .839; main effect prandial state p = .095; main effect mouth rinse solution p = .277). In line, mean power output and heart rate during exercise did not differ between trials. In conclusion, a sucrose mouth rinse does not improve ~1-hr time trial performance in well-trained cyclists when performed in either the fasted or the fed state.

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No Effect of Acute and 6-Day Nitrate Supplementation on VO2 and Time-Trial Performance in Highly Trained Cyclists

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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Daily Myofibrillar Protein Synthesis Rates in Response to Low- and High-Frequency Resistance Exercise Training in Healthy, Young Men

Brandon J. Shad, Janice L. Thompson, James Mckendry, Andrew M. Holwerda, Yasir S. Elhassan, Leigh Breen, Luc J.C. van Loon, and Gareth A. Wallis

The impact of resistance exercise frequency on muscle protein synthesis rates remains unknown. The aim of this study was to compare daily myofibrillar protein synthesis rates over a 7-day period of low-frequency (LF) versus high-frequency (HF) resistance exercise training. Nine young men (21 ± 2 years) completed a 7-day period of habitual physical activity (BASAL). This was followed by a 7-day exercise period of volume-matched, LF (10 × 10 repetitions at 70% one-repetition maximum, once per week) or HF (2 × 10 repetitions at ∼70% one-repetition maximum, five times per week) resistance exercise training. The participants had one leg randomly allocated to LF and the other to HF. Skeletal muscle biopsies and daily saliva samples were collected to determine myofibrillar protein synthesis rates using 2H2O, with intracellular signaling determined using Western blotting. The myofibrillar protein synthesis rates did not differ between the LF (1.46 ± 0.26%/day) and HF (1.48 ± 0.33%/day) conditions over the 7-day exercise training period (p > .05). There were no significant differences between the LF and HF conditions over the first 2 days (1.45 ± 0.41%/day vs. 1.25 ± 0.46%/day) or last 5 days (1.47 ± 0.30%/day vs. 1.50 ± 0.41%/day) of the exercise training period (p > .05). Daily myofibrillar protein synthesis rates were not different from BASAL at any time point during LF or HF (p > .05). The phosphorylation status and total protein content of selected proteins implicated in skeletal muscle ribosomal biogenesis were not different between conditions (p > .05). Under the conditions of the present study, resistance exercise training frequency did not modulate daily myofibrillar protein synthesis rates in young men.

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Carbohydrate Mouth Rinsing in the Fed State: Lack of Enhancement of Time-Trial Performance

Milou Beelen, Jort Berghuis, Ben Bonaparte, Sam B. Ballak, Asker E. Jeukendrup, and Luc J.C van Loon

It has been reported previously that mouth rinsing with a carbohydrate-containing solution can improve cycling performance. The purpose of the current study was to investigate the impact of such a carbohydrate mouth rinse on exercise performance during a simulated time trial in a more practical, postprandial setting. Fourteen male endurance-trained athletes were selected to perform 2 exercise tests in the morning after consuming a standardized breakfast. They performed an ~1-hr time trial on a cycle ergometer while rinsing their mouths with either a 6.4% maltodextrin solution (CHO) or water (PLA) after every 12.5% of the set amount of work. Borg’s rating of perceived exertion (RPE) was assessed after every 25% of the set amount of work, and power output and heart rate were recorded continuously throughout the test. Performance time did not differ between treatments and averaged 68.14 ± 1.14 and 67.52 ± 1.00 min in CHO and PLA, respectively (p = .57). In accordance, average power output (265 ± 5 vs. 266 ± 5 W, p = .58), heart rate (169 ± 2 vs. 168 ± 2 beats/min, p = .43), and RPE (16.4 ± 0.3 vs. 16.7 ± 0.3 W, p = .26) did not differ between treatments. Furthermore, after dividing the trial into 8s, no differences in power output, heart rate, or perceived exertion were observed over time between treatments. Carbohydrate mouth rinsing does not improve time-trial performance when exercise is performed in a practical, postprandial setting.

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Endocrine Responses During Overnight Recovery From Exercise: Impact of Nutrition and Relationships With Muscle Protein Synthesis

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.

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Acute Effects of Dietary Nitrate on Exercise Tolerance, Muscle Oxygenation, and Cardiovascular Function in Patients With Peripheral Arterial Disease

Cindy M.T. van der Avoort, Luc J.C. van Loon, Lex B. Verdijk, Paul P.C. Poyck, Dick T.J. Thijssen, and Maria T.E. Hopman

Previous studies have used supplements to increase dietary nitrate intake in clinical populations. Little is known about whether effects can also be induced through vegetable consumption. Therefore, the aim of this study was to assess the impact of dietary nitrate, through nitrate-rich vegetables (NRV) and beetroot juice (BRJ) supplementation, on plasma nitrate and nitrite concentrations, exercise tolerance, muscle oxygenation, and cardiovascular function in patients with peripheral arterial disease. In a randomized crossover design, 18 patients with peripheral arterial disease (age: 73 ± 8 years) followed a nitrate intake protocol (∼6.5 mmol) through the consumption of NRV, BRJ, and nitrate-depleted BRJ (placebo). Blood samples were taken, blood pressure and arterial stiffness were measured in fasted state and 150 min after intervention. Each intervention was followed by a maximal walking exercise test to determine claudication onset time and peak walking time. Gastrocnemius oxygenation was measured by near-infrared spectroscopy. Blood samples were taken and blood pressure was measured 10 min after exercise. Mean plasma nitrate and nitrite concentrations increased (nitrate; Time × Intervention interaction; p < .001), with the highest concentrations after BRJ (494 ± 110 μmol/L) compared with NRV (202 ± 89 μmol/L) and placebo (80 ± 19 μmol/L; p < .001). Mean claudication onset time and peak walking time did not differ between NRV (413 ± 187 s and 745 ± 220 s, respectively), BRJ (392 ± 154 s and 746 ± 176 s), and placebo (403 ± 176 s and 696 ± 222 s) (p = .762 and p = .165, respectively). Gastrocnemius oxygenation, blood pressure, and arterial stiffness were not affected by the intervention. NRV and BRJ intake markedly increase plasma nitrate and nitrite, but this does not translate to improved exercise tolerance, muscle oxygenation, and/or cardiovascular function.

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Endurance-Type Exercise Increases Bulk and Individual Mitochondrial Protein Synthesis Rates in Rats

Andrew M. Holwerda, Freek G. Bouwman, Miranda Nabben, Ping Wang, Janneau van Kranenburg, Annemie P. Gijsen, Jatin G. Burniston, Edwin C.M. Mariman, and Luc J.C. van Loon

Physical activity increases muscle protein synthesis rates. However, the impact of exercise on the coordinated up- and/or downregulation of individual protein synthesis rates in skeletal muscle tissue remains unclear. The authors assessed the impact of exercise on mixed muscle, myofibrillar, and mitochondrial protein synthesis rates as well as individual protein synthesis rates in vivo in rats. Adult Lewis rats either remained sedentary (n = 3) or had access to a running wheel (n = 3) for the last 2 weeks of a 3-week experimental period. Deuterated water was injected and subsequently administered in drinking water over the experimental period. Blood and soleus muscle were collected and used to assess bulk mixed muscle, myofibrillar, and mitochondrial protein synthesis rates using gas chromatography–mass spectrometry and individual muscle protein synthesis rates using liquid chromatography–mass spectrometry (i.e., dynamic proteomic profiling). Wheel running resulted in greater myofibrillar (3.94 ± 0.26 vs. 3.03 ± 0.15%/day; p < .01) and mitochondrial (4.64 ± 0.24 vs. 3.97 ± 0.26%/day; p < .05), but not mixed muscle (2.64 ± 0.96 vs. 2.38 ± 0.62%/day; p = .71) protein synthesis rates, when compared with the sedentary condition. Exercise impacted the synthesis rates of 80 proteins, with the difference from the sedentary condition ranging between −64% and +420%. Significantly greater synthesis rates were detected for F1-ATP synthase, ATP synthase subunit alpha, hemoglobin, myosin light chain-6, and synaptopodin-2 (p < .05). The skeletal muscle protein adaptive response to endurance-type exercise involves upregulation of mitochondrial protein synthesis rates, but it is highly coordinated as reflected by the up- and downregulation of various individual proteins across different bulk subcellular protein fractions.

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Dietary Protein Intake and Distribution Patterns of Well-Trained Dutch Athletes

Jenna B. Gillen, Jorn Trommelen, Floris C. Wardenaar, Naomi Y.J. Brinkmans, Joline J. Versteegen, Kristin L. Jonvik, Christoph Kapp, Jeanne de Vries, Joost J.G.C. van den Borne, Martin J. Gibala, and Luc J.C. van Loon

Dietary protein intake should be optimized in all athletes to ensure proper recovery and enhance the skeletal muscle adaptive response to exercise training. In addition to total protein intake, the use of specific proteincontaining food sources and the distribution of protein throughout the day are relevant for optimizing protein intake in athletes. In the present study, we examined the daily intake and distribution of various proteincontaining food sources in a large cohort of strength, endurance and team-sport athletes. Well-trained male (n=327) and female (n=226) athletes completed multiple web-based 24-hr dietary recalls over a 2-4 wk period. Total energy intake, the contribution of animal- and plant-based proteins to daily protein intake, and protein intake at six eating moments were determined. Daily protein intake averaged 108±33 and 90±24 g in men and women, respectively, which corresponded to relative intakes of 1.5±0.4 and 1.4±0.4 g/kg. Dietary protein intake was correlated with total energy intake in strength (r=0.71, p <.001), endurance (r=0.79, p <.001) and team-sport (r=0.77, p <.001) athletes. Animal and plant-based sources of protein intake was 57% and 43%, respectively. The distribution of protein intake was 19% (19±8 g) at breakfast, 24% (25±13 g) at lunch and 38% (38±15 g) at dinner. Protein intake was below the recommended 20 g for 58% of athletes at breakfast, 36% at lunch and 8% at dinner. In summary, this survey of athletes revealed they habitually consume > 1.2 g protein/kg/d, but the distribution throughout the day may be suboptimal to maximize the skeletal muscle adaptive response to training.