Recent work in humans suggests that increasing nitric oxide (NO) bioavailability may induce physiological changes beyond the well known hemodynamic effects (Dejam, Hunter et al. 2004; Webb, Patel et al. 2008). NO plays a key role in the regulation of blood flow, muscle contractility, myocyte differentiation, glucose and calcium homeostasis (Dejam, Hunter et al. 2004). Within the human body, exogenous nitrate (NO3-) which has a half-life of 6-7 hours (h) (Lundberg, Weitzberg et al. 2008) is reduced to bioactive nitrite (NO2-) by facultative anaerobic bacteria in the saliva and further to NO via various pathways (Duncan, Dougall et al. 1995; Zhang, Naughton et al. 1998). Several groups (Larsen, Weitzberg et al. 2007; Bailey, Fulford et al. 2009; Bailey, Winyard et al. 2010; Larsen, Weitzberg et al. 2010; Vanhatalo, Bailey et al. 2010; Lansley, Winyard et al. 2011) have investigated whether dietary nitrate provision affects metabolic or circulatory parameters during exercise in vivo in humans. Oral ingestion of sodium nitrate (0.1 mmol•kg-1•d-1) for 2-3 days (d) has been shown to significantly reduce pulmonary oxygen uptake (VO2) during submaximal cycling exercise in both untrained (Larsen, Weitzberg et al. 2010) and trained men (Larsen, Weitzberg et al. 2007). Since the use of sodium nitrate is regulated in most countries, researchers have started to examine the impact of ingesting nitrate-rich foods, such as beetroot juice, on the physiological response to exercise (Bailey, Fulford et al. 2009; Bailey, Winyard et al. 2010; Vanhatalo, Bailey et al. 2010; Lansley, Winyard et al. 2011).

Recent work by Jones and colleagues have demonstrated that ingestion of 0.5 L beetroot juice per d for 6 d reduces pulmonary oxygen uptake during submaximal exercise (Bailey, Fulford et al. 2009; Vanhatalo, Bailey et al. 2010; Lansley, Winyard et al. 2011) and lowers the ATP cost of muscle force production suggesting an enhanced contractile efficiency (Bailey, Winyard et al. 2010). This improvement in exercise efficiency was evident acutely (2.5 h) after ingestion of a single 0.5 L bolus of beetroot and persisted for 15 d when supplementation was continued (Vanhatalo, Bailey et al. 2010). Although the active ingredient in beetroot has been assumed to be nitrate, beetroot is rich in several other potentially metabolically-active compounds (e.g. polyphenols). To confirm whether the proposed cardiovascular and physiological benefits of beetroot juice are solely attributable to its high NO3- content, Jones and colleagues tested beetroot juice against nitrate-depleted beetroot juice. They verified their previous findings (Bailey, Fulford et al. 2009) by demonstrating a lowered O2 cost of submaximal exercise following ingestion of 0.5 L beetroot juice for 6 d when compared with the nitrate-depleted juice (Lansley, Winyard et al. 2011).

From the proposed improvements in metabolic efficiency, it has been suggested that nitrate supplementation (0.5 L•d-1; \~5.1-11.2 mmol NO3- •d-1) may increase exercise tolerance or time to fatigue when exercise is performed at higher workloads (Bailey, Fulford et al. 2009; Bailey, Winyard et al. 2010; Vanhatalo, Bailey et al. 2010; Lansley, Winyard et al. 2011). The latter suggests that inorganic nitrate ingestion may act as a strong ergogenic aid. We recently tested the potential ergogenic properties of nitrate using a more practical, performance-based study that simulated athletic competition in a trained subject population (Cermak, Gibala et al. 2011). We demonstrated that following 6 d of dietary nitrate supplementation in the form of concentrated beetroot juice (140 mL•d-1; \~4 mmol NO3- •d-1), mean VO2 was lower during submaximal exercise and 10 km time-trial performance improved in trained cyclists (Cermak, Gibala et al. 2011). However, the minimal dosage and duration of nitrate supplementation that is needed to elicit these performance effects remains largely unknown. Although time to exhaustion is not a very practical performance measurement, improvements in such performance tests have been observed after only 4 d of dietary nitrate supplementation (0.5 L•d-1; \~6.2 mmol•d-1 NO3-). Furthermore, even a single dose of nitrate (\~5.2 mmol NO3-) has been shown to lower mean VO2 values measured 2.5 h after ingesting 0.5 L of beetroot juice (Vanhatalo, Bailey et al. 2010). Whether a similar improvement in time-trial performance would also be observed after a shorter supplementation period is presently unknown. Therefore, the present study aims to investigate whether a single dose of dietary nitrate (140 mL; \~8 mmol NO3-) ingested 3 h prior to the onset of exercise will improve time-trial performance in trained cyclists.

Furthermore, inorganic nitrate ingestion (beetroot juice) has also been demonstrated to improve time to claudication pain while walking in peripheral artery disease patients (Kenjale, Ham et al. 2011), and lower plasma triglycerides in patients at risk for cardiovascular disease (Zand, Lanza et al. 2011), providing evidence for inorganic nitrate ingestion to improve vasodilation/perfusion. Whether a bolus ingestion of nitrate would also influence the perfusion of nutrients following the ingestion of a small meal remains unknown. Therefore, in a secondary aim, we want to investigate the plasma metabolite response in addition to nitrate and nitrite concentrations following the nitrate supplementation and subsequent meal ingestion.

1. OBJECTIVES The main objective will be to identify whether a bolus (140 mL) ingestion of dietary nitrate (\~8 mmol NO3-) in the form of concentrated beetroot juice will acutely increase time-trial performance compared to a nitrate-depleted placebo (140 mL; \~0.0047 mmol NO3-). A secondary objective will be to measure blood samples after nitrate and meal ingestion to assess any changes in the concentration of nitrate, nitrite, glucose, insulin, lactate and free fatty acids. In this secondary objective, we are most interested in determining whether nitrate supplementation changes blood metabolites (glucose, insulin, lactate, free fatty acids) following 1) meal ingestion and 2) immediate post-exercise recovery period in comparison to the placebo. We will test the hypothesis that a single dose (140 mL; \~8 mmol NO3-) of dietary nitrate supplementation (beetroot juice) ingested 3 h prior to exercise will improve time-trial performance in trained cyclists compared to the nitrate-depleted placebo.
2. STUDY DESIGN (Protocol) After the assessment of aerobic capacity (step-wise exercise cycling test to exhaustion) and familiarization testing, subjects will be randomized in a double-blind fashion to the treatment order of a bolus ingestion of nitrate (beetroot juice) and placebo (nitrate-depleted beetroot juice). For the two experimental trials, subjects will report to the laboratory at 8.00 h for the insertion of the blood catheter into an antecubital vein. Subjects will then be asked to consume 140 mL of either concentrated beetroot juice nitrate or concentrated nitrate-depleted beetroot juice (placebo). Following the consumption of the beverage, subjects will be given a standardized breakfast and will then rest in the lab for 2.5 h before being weighed and fitted with a heart-rate monitor for the commencement of the time-trial (3 h post ingestion of treatment beverage). Blood samples will be drawn at time 0 min (before beverage ingestion), 30, 60, 90, 120, 150, 180 min (start of time-trial). Two more blood samples will be drawn immediately post and 30 minutes post time-trial (Figure 1). Sampling will occur every 30 minutes based on previous research examining blood metabolites following meal ingestion or an oral glucose load (van Dijk, Manders et al. 2011) and nitrate/nitrite concentrations (Webb, Patel et al. 2008). The last two blood samples will occur immediately following the time-trial and 30 min into the recovery period to assess any changes in metabolites and nitrate/nitrite concentrations from the exercise and during the short-term recovery period.

To assess time-trial performance, subjects will be instructed to perform a set amount of work in the shortest time possible. Total work to be performed will be calculated according to the equation of Jeukendrup, Saris, Brouns, and Kester (Jeukendrup, Saris et al. 1996), adapted by our lab (Beelen, Berghuis et al. 2009) :

Total amount of work = 0.60 • Wmax • 3,600

where Wmax is the maximal workload capacity determined during Visit 1 and 3,600 is the duration in seconds (equivalent to 1 hr). The ergometer will be set in linear mode to obtain 60% Wmax when subjects' cycle at their preferred cadence determined during Visit 1. The ergometer will be connected to a computer that will calculate and display the total amount of work performed. Subjects will receive no verbal or physiological feedback during the time-trial. The only information subjects will receive is the absolute amount of work performed and the percentage of total work performed relative to the set amount of work that needs to be completed. A fan will be placed 1 meter behind each participant to provide cooling and air circulation during the trials. Heart rate (Polar, Finland) will be recorded continuously throughout the test. This type of time-trial has been validated and used before in our lab (Beelen, Berghuis et al. 2009), for an overview of validation studies see (Currell and Jeukendrup 2008). Examples of coefficients of variation in similar trials are 1.1 (Palmer, Dennis et al. 1996), 0.7 (Smith, Davison et al. 2001) and 0.9 (Laursen, Shing et al. 2003). Water will be provided ad libitum during visits 1 and 2. However, the water consumed during visit 2 (familiarization trial) will be measured and repeated for visits 3 and 4 (exercise trials).