Subjects. Ten healthy, recreationally active men (mean ± SD: age 19 ± 1 yr, height 1.80 ± 0.08 m, body mass 79 ± 11 kg) volunteered to participate in the study. None of the subjects smoked tobacco or used dietary supplements. The procedures employed in the study were approved by the Institutional Research Ethics Committee. All subjects gave their written informed consent prior to the commencement of the study, after the experimental procedures, associated risks, and potential benefits of participation had been explained. Subjects were instructed to arrive at the laboratory in a rested and fully hydrated state, ≥3 h postprandial, and to avoid strenuous exercise in the 24 h preceding each testing session. Each subject was also asked to abstain from caffeine and alcohol for 6 and 24 h, respectively, before each test. All tests were performed at the same time of day (±2 h).

Experimental design. Subjects were required to report to the laboratory on eight occasions over 6–7 wk to complete the experimental testing. On the first visit to the laboratory, subjects completed a ramp incremental exercise test for determination of the gas exchange threshold (GET) and peak V̇o 2 (V̇o 2 peak ). Subjects were familiarized with the two exercise performance tests employed in the study during the second laboratory testing session. After these preliminary exercise tests, subjects returned to the laboratory on days 6 and 7 of 7-day supplementation periods with Pla, Arg, and Cit to complete the experimental testing. During these tests, resting blood pressure, pulmonary V̇o 2 kinetics, muscle oxygenation, and exercise performance were assessed, and a resting venous blood sample was obtained. The supplements were administered orally in a randomized order as part of a double-blind, crossover experimental design. Each supplementation period was separated by 7–10 days of washout. A food diary was provided for the first supplementation intervention, and the subjects were instructed to replicate their diet over subsequent supplementation periods.

Incremental test. During the first laboratory visit, subjects completed a ramp incremental cycle test on an electronically braked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands). Initially, subjects performed 3 min of baseline cycling at 0 W; then the work rate was increased by 30 W/min until the limit of tolerance. The subjects cycled at a self-selected pedal rate (70–90 rpm), which, along with saddle and handle bar heights and configuration, was recorded and reproduced in subsequent tests. Breath-by-breath pulmonary gas-exchange data were collected continuously during the incremental tests and averaged over consecutive 10-s periods. The maximum V̇o 2 (V̇o 2 max ) was taken as the highest 30-s mean value attained prior to the subject's volitional exhaustion in the test. The GET was determined from a cluster of measurements, including 1) the first disproportionate increase in CO 2 production (V̇co 2 ) from visual inspection of individual plots of V̇co 2 vs. V̇o 2 , 2) an increase in expired ventilation (V̇e)/V̇o 2 with no increase in V̇e/V̇co 2 , and 3) an increase in end-tidal Po 2 with no fall in end-tidal Pco 2 . The work rates that would require 90% of the GET (moderate-intensity exercise) and 70%Δ (GET + 70% of the difference between the work rate at the GET and V̇o 2 max ; severe-intensity exercise) were subsequently calculated with account taken of the mean response time (MRT) for V̇o 2 during ramp exercise (i.e., two-thirds of the ramp rate was deducted from the work rate at GET and peak).

Familiarization tests. To avoid any order effect on the performance results as a consequence of a potential “learning effect,” subjects were familiarized with all performance tests prior to the experimental testing. Subjects completed a severe-intensity step exercise test terminating with an all-out sprint (exercise performance test) followed, after a 45-min passive recovery period, by a severe-intensity constant-work-rate step exercise test that was continued until the limit of tolerance (exercise tolerance test).

Supplementation procedures. Experimental testing was conducted during a 7-day supplementation period with Pla, Arg, and Cit. The Pla supplement consisted of 10.7 g of maltodextrin, the Arg supplement consisted of 6 g of Arg + 4.3 g of maltodextrin, and the Cit supplement consisted of 6 g of Cit + 4.3 g of maltodextrin. All supplements were energy-matched, containing 40 kcal per serving. Pure maltodextrin, Arg, and Cit powders (NOW Sports Nutrition, NOW Foods, Bloomingdale, IL) were mixed with 500 ml of water and 75 ml of black currant cordial in the proportions described above to produce the Pla, Arg, and Cit supplements. On days 1–5 of supplementation, subjects were instructed to drink the beverage slowly over the course of the day. On days 6 and 7 of supplementation, subjects were instructed to consume the beverage over a 10-min window, such that the entire beverage had been consumed 60 min before the subject was required to report to the laboratory.

Experimental tests. After reporting to the laboratory on days 6 and 7 of the supplementation interventions, subjects were required to rest in a seated position for 10 min in an isolated room. Thereafter, while the subject was seated, blood pressure of the brachial artery was measured using an automated sphygmomanometer (Dinamap Pro, GE Medical Systems, Tampa, FL). Four measurements were taken, and the mean of the measurements was calculated. A venous blood sample was then drawn into a lithium-heparin tube and centrifuged at 4,000 rpm and 4°C for 10 min, within 3 min of collection. Plasma was subsequently extracted and immediately frozen at −80°C for later analysis of [NO 2 −] in duplicate via chemiluminescence (6), and [Arg], [Cit], and l-ornithine (Orn) concentration ([Orn]) were determined using high-performance liquid chromatography (HPLC; see below). At 30 min after arrival at the laboratory (90 min after ingestion of the supplement), subjects completed a series of cycle exercise tests. We elected to commence exercise testing 90 min after supplement consumption, since published pharmacokinetic data have shown that this time frame should coincide with peak plasma [Arg] after oral ingestion of 6 g of Cit (53) or 6 g of Arg (10). The exercise protocol consisted of three “step” exercise tests: two moderate-intensity step tests followed by one severe-intensity exercise bout. Moderate-intensity step tests were completed to assess V̇o 2 kinetics and cycling economy in the absence of a V̇o 2 slow component, while severe-intensity step tests were completed to assess V̇o 2 kinetics in the presence of a V̇o 2 slow component, where V̇o 2 max is attained and the tolerable duration of exercise is <20 min (49, 66). We conducted repeated step tests on the same laboratory visit, since a prior moderate-intensity step exercise bout does not affect V̇o 2 kinetics during subsequent moderate- or severe-intensity cycle exercise (12, 17). Therefore, all subjects performed a total of four bouts of moderate-intensity exercise and two bouts of severe-intensity exercise for each experimental condition. Each transition began with 3 min of baseline cycling at 20 W before an abrupt transition to the target work rate. A passive recovery of 5 min separated the transitions. The duration of each moderate-intensity step was 6 min. On day 6 of each supplementation condition, subjects cycled for 6 min at a severe-intensity constant work rate (70%Δ) followed immediately by a 60-s all-out sprint. The resistance on the pedals during the 60-s all-out effort was set using the linear mode of the Lode ergometer, so that the subject would attain the power output calculated to be 50%Δ if he attained his preferred cadence (linear factor = power/preferred cadence2). Subjects were provided with a 5-s countdown prior to the sprint and were instructed to attain the peak power as quickly as possible and to continue exercising maximally for the duration of the sprint. No time feedback was given to the subjects at any point during the sprint. On day 7 of the supplementation period, the severe-intensity constant-work-rate bout was continued to the limit of tolerance. The time to task failure was used as a measure of exercise tolerance and was recorded when the pedal rate fell by >10 rpm below the required pedal rate.

Measurements. During all tests, pulmonary gas exchange and ventilation were measured breath-by-breath, with subjects wearing a nose clip and breathing through a low-dead-space, low-resistance mouthpiece-and-impeller turbine assembly (Triple V, Jaeger, Hoechberg, Germany). The inspired and expired gas volume was continuously sampled at 100 Hz; gas concentration signals were continuously sampled at 100 Hz using paramagnetic (O 2 ) and infrared (CO 2 ) analyzers (Oxycon Pro, Jaeger) via a capillary line connected to the mouthpiece. The gas analyzers were calibrated before each test with gases of known concentration, and the turbine volume transducer was calibrated with a 3-liter syringe (Hans Rudolph, Kansas City, MO). The volume and concentration signals were time-aligned by accounting for the delay in the capillary gas transit and the analyzer rise time relative to the volume signal. Pulmonary gas exchange and ventilation were calculated and displayed breath-by-breath. During the exercise trials, a blood sample was collected from a fingertip into a capillary tube over the 20 s preceding the step transition in work rate, the 20 s preceding the completion of 360 s of moderate- and severe-intensity cycling exercise, and immediately following the all-out sprint and immediately after exhaustion during the severe-intensity constant-work-rate trial. These whole blood samples were subsequently analyzed to determine blood lactate concentration ([lactate]; YSI 1500, Yellow Springs Instruments, Yellow Springs, OH) within 30 s of collection. The oxygenation status of the vastus lateralis of the right leg was monitored using a commercially available near-infrared spectroscopy (NIRS) system (model NIRO 300, Hamamatsu Photonics KK, Hiugashi-ku, Japan). The system consisted of an emission probe that irradiates laser beams and a detection probe. Four different-wavelength (776, 826, 845, and 905 nm) laser diodes provided the light source, and the light returning from the tissue was detected by a photomultiplier tube in the spectrometer. The intensity of incident and transmitted light was recorded continuously at 2 Hz and used to estimate concentration changes from the resting baseline for oxygenated, deoxygenated, and total tissue hemoglobin/myoglobin. Therefore, the NIRS data represent a relative change based on the optical density measured in the first datum collected. The deoxygenated hemoglobin/myoglobin concentration ([HHb]) signal was assumed to provide an estimate of changes in fractional O 2 extraction in the field of interrogation (21). It should be noted here that the contribution of deoxygenated myoglobin to the NIRS signal is unclear, and, as such, the terms [HbO 2 ] and [HHb] should be considered to refer to the combined concentrations of oxygenated and deoxygenated hemoglobin/myoglobin, respectively. The tissue oxygenation index (TOI) was calculated using the following equation TOI = [ HbO 2 ] [ HbO 2 ] + [ HHb ] × 100 (1) The leg was initially cleaned and shaved around the belly of the muscle, and the optodes were placed in the holder, which was secured to the skin with adhesive at 20 cm above the fibular head. To secure the holder and wires in place, an elastic bandage was wrapped around the subject's leg. The wrap helped minimize the possibility that extraneous light could influence the signal and also ensured that the optodes did not move during exercise. Indelible pen marks were made around the holder to enable precise reproduction of the placement in subsequent tests. The probe gain was set with the subject at rest in a seated position with the leg extended at down stroke on the cycle ergometer before the first exercise bout, and NIRS data were collected continuously throughout the exercise protocols. The data were subsequently downloaded onto a personal computer, and the resulting text files were stored on disk for later analysis. Plasma [Arg], [Cit], and [Orn] were determined by o-phthaldialdehyde (OPA)-derivatized, fluorescence-detection HPLC according to methods adapted from Jones and Gilligan (27). The HPLC apparatus was a Flexar LC system with Chromera software (Perkin Elmer). Briefly, plasma was deproteinized in 1.5 N perchloric acid, neutralized in 2 N potassium hydrogen carbonate, and centrifuged; then 100 μl of supernatant, 100 μl of 1.2% benzoic acid, and 1.4 ml of water were added to HPLC vials, and 50 μl of unknowns/standards were mixed with 50 μl of an OPA solution containing 2-mercaptoethanol (Fluoraldehyde OPA reagent solution, Thermo Scientific), enabling the precolumn derivatization of amino acids with a highly fluorescent OPA adduct. Twenty-five microliters of derivatized sample were mixed in mobile phase and eluted at 0.8 ml/min through a 4.6 × 150 mm, 2.7-μm Brownlee SPP C18 reverse-phase analytical column with 5-mm guard column with matching specification. A gradient protocol of aqueous mobile phase A (0.05 M potassium phosphate buffer, pH 7.2) with organic mobile phase B (40:40:20 acetonitrile-methanol-water) was performed: 0–1.5 min, 80% mobile phase A; 1.5–18.5 min, 80-65%; 23.5 min, 50%; 32.5 min, 40%; 36.5 min, 30%; 43.5 min, 0%; 51.5 min, 80%. Fluorescence was monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Amino acid concentrations were determined against standard calibration curves between 0 and 500 μM (nmol/ml).

Data analysis procedures. The breath-by-breath V̇o 2 data from each test were initially examined to exclude errant breaths caused by coughing, swallowing, sighing, etc., and values >4 SDs from the local mean were removed. The breath-by-breath data were subsequently linearly interpolated to provide second-by-second values, and, for each individual, identical repetitions were time-aligned to the start of exercise and ensemble-averaged. The first 20 s of data after the onset of exercise (i.e., the phase I response) were deleted, and a nonlinear least-squares algorithm was used to fit the data thereafter. A single-exponential model was used to characterize the V̇o 2 responses to moderate exercise, and a biexponential model was used for severe exercise, as described in the following equations V ˙ O 2 ( t ) = V ˙ O 2 baseline + A p ( 1 − e − ( t − TD p / τp ) ) ( moderate ) (2) V ˙ O 2 ( t ) = V ˙ O 2 baseline + A p ( 1 − e − ( t − TD p / τp ) ) + A s ( 1 − e − ( t − TD s / τs ) ) ( severe ) (3) where V̇o 2 (t) represents the absolute V̇o 2 at a given time t; V̇o 2baseline represents the mean V̇o 2 in the baseline period; A p , TD p , and τ p represent the amplitude, time delay, and time constant, respectively, describing the phase II increase in V̇o 2 above baseline; and A s , TD s , and τ s represent the amplitude of the V̇o 2 slow component, time delay before the onset of the V̇o 2 slow component, and time constant describing the development of the V̇o 2 slow component, respectively. An iterative process was used to minimize the sum of the squared errors between the fitted function and the observed values. V̇o 2baseline was defined as the mean V̇o 2 measured over the final 90 s of the resting baseline period. The V̇o 2 at 360 s was taken as the mean V̇o 2 between 330 and 360 s, while the V̇o 2 at the limit of tolerance (T lim ) was defined as the mean V̇o 2 measured over the final 30 s of the exhaustive exercise bout. Because the asymptotic value (A s ) of the exponential term describing the V̇o 2 slow component may represent a higher value than is actually reached at the end of the exercise, the actual amplitude of the V̇o 2 slow component at the end of exercise was defined as A s ′. The A s ′ parameter was compared at the same isotime (360 s) for all dietary interventions. The amplitude of the slow component was also described relative to the entire V̇o 2 response. In addition, the functional “gain” of the fundamental V̇o 2 response was computed by dividing A p by the Δwork rate. To determine the overall kinetics of the V̇o 2 response to moderate- and severe-intensity exercise, the data were also fit with a monoexponential model from 0 s to end exercise without time delay. This MRT was used to calculate the O 2 deficit using the following equation O 2 Deficit ( L ) = MRT ( min ) × Δ V ̇ O 2 ( L ) (4) where ΔV̇o 2 is the difference in V̇o 2 between 360 s and baseline. To provide information on muscle oxygenation, we also modelled the [HHb] response to exercise. Mono- and biexponential models, similar to those described above, were applied to the ensemble-averaged data, with the exception that the fitting window commenced at the time at which the [HHb] signal increased 1 SD above the baseline mean. The [HHb] kinetics for moderate exercise were determined by constraining the fitting window to the point at which monoexponentiality became distorted, consequent to a gradual fall in [HHb], as determined by visual inspection of the residual plots. The [HHb] kinetics for severe exercise were determined by fitting a biexponential model from the first data point, which was 1 SD above the baseline mean through the entire response. The [HHb] TD and τ values were summed to provide information on the overall [HHb] response dynamics in the fundamental phase of the response. The [HbO 2 ] response does not approximate an exponential and was, therefore, not modelled. Rather, we assessed this by determining the [HbO 2 ] at baseline (90 s preceding step transition), 120 s (30-s mean surrounding 120 s), and end exercise (mean response over the final 30 s of exercise). The TOI responses were assessed using the same data analysis procedures.