Influence of dietary nitrate supplementation on physiological and muscle metabolic adaptations to sprint interval training

We investigated the influence of nitrate-rich and nitrate-depleted beetroot juice on the muscle metabolic and physiological adaptations to 4 wk of sprint interval training. Compared with placebo, dietary nitrate supplementation reduced the O₂ cost of submaximal exercise, resulted in greater improvement in incremental (but not severe-intensity) exercise performance, and augmented some muscle metabolic adaptations to training. Nitrate supplementation may facilitate some of the physiological responses to sprint interval training.

Abstract

We hypothesized that 4 wk of dietary nitrate supplementation would enhance exercise performance and muscle metabolic adaptations to sprint interval training (SIT). Thirty-six recreationally active subjects, matched on key variables at baseline, completed a series of exercise tests before and following a 4-wk period in which they were allocated to one of the following groups: 1) SIT and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-depleted beetroot juice as a placebo (SIT+PL); 2) SIT and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice (~13 mmol $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$/day; SIT+BR); or 3) no training and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice (NT+BR). During moderate-intensity exercise, pulmonary oxygen uptake was reduced by 4% following 4 wk of SIT+BR and NT+BR (P < 0.05) but not SIT+PL. The peak work rate attained during incremental exercise increased more in SIT+BR than in SIT+PL (P < 0.05) or NT+BR (P < 0.001). The reduction in muscle and blood [lactate] and the increase in muscle pH from preintervention to postintervention were greater at 3 min of severe-intensity exercise in SIT+BR compared with SIT+PL and NT+BR (P < 0.05). However, the change in severe-intensity exercise performance was not different between SIT+BR and SIT+PL (P > 0.05). The relative proportion of type IIx muscle fibers in the vastus lateralis muscle was reduced in SIT+BR only (P < 0.05). These findings suggest that BR supplementation may enhance some aspects of the physiological adaptations to SIT.

NEW & NOTEWORTHY We investigated the influence of nitrate-rich and nitrate-depleted beetroot juice on the muscle metabolic and physiological adaptations to 4 wk of sprint interval training. Compared with placebo, dietary nitrate supplementation reduced the O₂ cost of submaximal exercise, resulted in greater improvement in incremental (but not severe-intensity) exercise performance, and augmented some muscle metabolic adaptations to training. Nitrate supplementation may facilitate some of the physiological responses to sprint interval training.

the gaseous biological signaling molecule, nitric oxide (NO), is known to modulate several physiological responses to exercise including skeletal muscle perfusion, energy metabolism, and contractile function (41, 69). Nitric oxide synthase enzymes catalyze the oxygen (O₂)-dependent production of NO from l-arginine, and it is now known that the products of NO oxidation, nitrate ($\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$) and nitrite ($\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$), can be reduced in vivo to form NO (52, 72). Interestingly, hypoxia and acidosis, physiological environments typical of muscular exercise, facilitate the reduction of $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ to NO (72). Increasing the dietary intake of inorganic $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ to augment circulating $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ and $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ pools may therefore represent a natural means to increase NO bioavailability during exercise.

The physiological effects of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ ingestion in humans are well documented and may include a reduction in blood pressure (BP) at rest and reduced oxygen uptake (V̇o₂) during submaximal exercise (5, 16, 45, 73). Moreover, several studies suggest that $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation can improve performance in a variety of exercise settings, at least in subelite athletes (2, 14, 71, 73, 78, cf. 40). It has recently been reported that short-term (3–7 days) $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation may favorably impact the metabolic and contractile properties of skeletal muscle (30, 46, 76). Specifically, the improvements in exercise efficiency and performance that have been observed following dietary $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation may be related to altered mitochondrial function (46, cf. 76) and to enhanced muscle force or power production (19, 30), which, in turn, might be related to increased perfusion and contractile function (22, 32). It is unclear whether more protracted periods (several weeks) of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation may more favorably impact the physiological response to exercise and improve exercise performance. However, given that dietary $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ may specifically enhance the physiological responses of type II muscle fibers to exercise (22, 23, 32, 35) and improve performance during repeated sprint exercise (2, 71, 78), it is possible that $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation may be of particular value to athletes engaging in high-intensity training.

Sprint interval training (SIT) is known to provide a potent and relatively time-efficient stimulus for enhancing aerobic capacity and endurance exercise performance (11–13, 26). However, the effects of a high-$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ dietary supplement, such as beetroot juice, consumed daily as part of an exercise-training program, on the physiological and muscle metabolic adaptations to training have received limited attention (21, 57). It is possible that the NO-mediated inhibition of O₂ consumption at cytochrome c oxidase (10, 17) and resultant local hypoxia may initiate signaling cascades that may be synergistic (or antagonistic) to those generated by SIT (27). Also, similar to the effects of training, elevated NO bioavailability may stimulate angiogenesis (25), mitochondrial biogenesis (58), and the transformation of muscle fiber phenotype (59, 68) through cGMP-dependent gene expression and the activation of regulatory factors, in particular peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α; 37, 43). It could also be anticipated that the lower V̇o₂ and reduced adenosine triphosphate (ATP) and phosphocreatine (PCr) cost of muscle force production during high-intensity exercise following $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation (6, 24) might enable a higher training intensity for the same effort, which, over time, may lead to greater training adaptation (34). An increase in cytosolic calcium concentration ([Ca²⁺]) and force production during muscle contraction following $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation (32) may also permit a higher training intensity to be maintained. Given the potentially complementary effects of exercise training and NO bioavailability on metabolic regulation, it is possible that $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation could augment the physiological adaptations to SIT.

Two recent studies have used different approaches to address this question and have produced somewhat disparate results (21, 57). Muggeridge et al. (57) reported that compared with placebo, supplementation with $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-containing gels during 3 wk of SIT (4–6 × 15-s sprints, 3 times per week) tended to increase peak work rate during incremental exercise (8.7 vs. 4.7%; P = 0.07) and reduce the fatigue index during repeated sprint exercise (0.5 vs. 7.3%; P = 0.06). De Smet et al. (21) reported that compared with placebo, NaNO₃ supplementation during 5 wk of SIT (4–6 × 30-s sprints, 3 times per week), performed in hypoxia, did not improve either incremental exercise or 30-min time trial performance but did result in a significant increase in the proportion of type IIa fibers in the vastus lateralis muscle. Neither study measured potential training-related differences in muscle metabolic responses to exercise with $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ compared with placebo supplementation (for example, [PCr], pH, [lactate], and [glycogen] as determined from muscle biopsy) or compared the effects of training with $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ or placebo with the physiological effects of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation alone. It would be of interest to determine whether the intriguing change in muscle fiber type proportions when SIT in hypoxia was performed with $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation (21) is also evident following SIT in normoxia. Additional studies are required to explore the influence of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation on the muscle metabolic adaptations and submaximal and maximal exercise responses to training.

The purpose of this study was, therefore, to evaluate the independent and combined performance and physiological effects of SIT and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation during a 4-wk intervention involving the following: SIT with concurrent $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-depleted beetroot juice supplementation as a placebo (PL); SIT with concurrent $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice supplementation (BR); and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice supplementation with no training. We tested the hypothesis that 4-wk SIT and 4-wk BR supplementation would independently improve physiological responses and exercise performance but that these effects would be greater when BR supplementation and SIT were combined.

METHODS

Subjects

Eighteen male (age 27 ± 8 yr, height 1.79 ± 0.08 m, body mass 80 ± 13 kg, V̇o_2peak 50.4 ± 11.4 ml·kg⁻¹·min⁻¹; means ± SD) and 18 female (age 23 ± 4 yr, height 1.66 ± 0.05 m, body mass 65 ± 9 kg, V̇o_2peak 39.8 ± 5.8 ml·kg⁻¹·min⁻¹) participants were recruited. The subjects were recreationally active sportspeople involved in team and/or endurance sports, but they were not highly trained. Following an explanation of the experimental procedures, associated risks, potential benefits, and likely value of the possible findings, subjects gave their written informed consent to participate. The study was approved by the Institutional Research Ethics Committee and conformed to the code of ethics of the Declaration of Helsinki.

Experimental Design

Subjects initially visited the laboratory on three separate occasions over a 5-day period. On visit 1, subjects performed an incremental exercise test on a cycle ergometer for the determination of V̇o_2peak and gas exchange threshold (GET)_. The work rates requiring 80% of the GET (moderate exercise) and 85% Δ (GET plus 85% of the difference between the work rate at GET and V̇o_2peak; severe exercise) were calculated and adjusted for mean response time for V̇o₂ during incremental exercise (75). Following this, subjects were familiarized to the exercise testing procedures, including completion of a severe-intensity bout of cycle ergometry until exhaustion. On visit 2, subjects completed a 5-min bout of moderate-intensity cycling and an incremental exercise test. On visit 3, subjects completed two bouts of severe-intensity cycling, the first for 3 min and the second until task failure.

In a double-blind, independent-groups design, subjects were then assigned to receive $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice (BR) or $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-depleted beetroot juice (PL) for 28 days. Three independent groups (n = 12, comprising 6 men and 6 women) were matched at baseline for physical characteristics (i.e., mass, height, and age) as well as physiological and performance variables of interest, principally BP and peak work rate (WR) during incremental exercise and, secondarily, V̇o_2peak and GET. Subjects were then either enrolled into a 4-wk supervised SIT program with PL (SIT+PL) or BR (SIT+BR) supplementation or received the $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice for 28 days without undergoing a training intervention (NT+BR).

All groups completed the same exercise tests (at the same absolute work rates) and physiological assessments both before and after the 28-day intervention period. Also, after 14 days, subjects visited the laboratory for an incremental exercise test to assess the short-term changes in aerobic capacity that may be expected following the interventions (11, 65, 73).

Laboratory visits were scheduled at the same time of day (±2 h). Subjects were asked to maintain their normal dietary and exercise behavior throughout the study. However, subjects were instructed to record their diet during the 24 h preceding the first laboratory visit and to repeat this for all subsequent laboratory visits. On days of training, subjects were asked to arrive at the training venue ≥1 h postprandial and to complete a 5-min self-paced warm-up before training commenced. On experimental days, subjects were instructed to arrive at the laboratory ≥3 h postprandial having avoided strenuous exercise and the consumption of alcohol and caffeine in the 12 h preceding each exercise test. For the duration of the study, subjects were asked to refrain from taking other dietary supplements and also to avoid using antibacterial mouthwash as this inhibits the reduction of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ to $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ in the oral cavity by eliminating commensal bacteria (29).

Supplementation.

Following the preintervention laboratory visits, subjects were allocated to receive concentrated $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice (BR; beetroot juice; ~6.4 mmol of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ per 70 ml; Beet it; James White Drinks, Ipswich, United Kingdom) or $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-depleted beetroot juice (PL; placebo beetroot juice; ~0.04 mmol $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ per 70 ml; Beet it; James White Drinks). Subjects consumed 1 × 70 ml of their allocated supplement each morning and evening for the duration of the training or nontraining intervention and recorded their intake in a diary. This approach would be expected to result in elevated plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] and [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] for each 24-h period (79). Compliance was checked by the return of empty bottles each week and via questionnaire at 2 and 4 wk. BR and PL doses were administered using a double-blind design. On experimental visits at the midintervention point and following the intervention period, subjects consumed 2 × 70 ml of their allocated supplement 2.5 h before the exercise tests.

Incremental exercise tests.

On the first laboratory visit before and following the intervention period as well as at the midintervention point, subjects completed a ramp incremental exercise test on an electronically braked cycle ergometer (Lode Excalibur Sport; Groningen, The Netherlands). The self-selected cadence (75–90 rpm) and saddle and handlebar height and configuration for each subject were recorded on the first visit and reproduced in subsequent visits. Initially, subjects performed 3 min of baseline cycling at 20 W, after which the work rate was increased by 30 W/min until the limit of tolerance. Breath-by-breath pulmonary gas exchange data (Oxycon Pro; Jaeger, Höchberg, Germany) were collected continuously throughout all incremental tests and were averaged over 10-s periods. V̇o_2peak and GET were determined as previously described (73).

Step exercise tests.

A 5-min moderate-intensity “step” test was performed on the first laboratory visit before and following the intervention. This was completed 10 min before the ramp incremental test protocol was initiated. On the second laboratory visit before and following the intervention, two severe-intensity step tests were performed, separated by a 20-min period of rest; the first until 3 min, and the second, after 20 min of passive recovery, until task failure. The time to task failure was recorded once the pedal rate fell by >10 rpm below the target cadence. All step tests began with 3 min of pedaling at 20 W before a sudden transition to the target work rate. Muscle biopsies were obtained before and following the 3-min severe-intensity exercise bout and again at task failure in the second bout. Breath-by-breath pulmonary gas exchange data were collected continuously throughout all step tests.

Training intervention.

Following the initial laboratory visits, subjects were allocated to one of the two SIT groups: SIT with PL supplementation (SIT+PL; age 25 ± 7 yr, height 174 ± 10 cm, body mass 73 ± 10 kg); SIT with BR supplementation (SIT+BR; age 24 ± 7 yr, height 174 ± 11 cm, body mass 78 ± 18 kg); or the nontraining group with BR supplementation (NT+BR; age 25 ± 7 yr, height 170 ± 6 cm, body mass 68 ± 9 kg). All three groups consisted of six male and six female subjects. Both SIT groups completed a total of 14 supervised training sessions over a 4-wk period, with at least 24 h separating each training session, while the NT group maintained their habitual exercise patterns. The postintervention laboratory tests were performed at least 48 h following, but within 4 days of, completing the final training session.

During the training sessions, the SIT groups completed a series of 30-s “all-out” sprints (i.e., Wingate test) against a resistance equivalent to 7.5% body mass on a mechanically braked ergometer (model 814E bicycle ergometer; Monark, Stockholm, Sweden; 11–13). Each sprint was separated by a 4-min period of rest in which subjects cycled at a low cadence against a light resistance to reduce venous pooling and sensations of nausea. During weeks 1 and 2 of training, subjects performed 4 × 30-s sprints three times per week, while during weeks 3 and 4, subjects performed 5 × 30-s sprints four times per week. Following a 5-min warm-up of cycling against a light resistance, subjects were given a 10-s countdown and instructed to pedal maximally for 2 s before the appropriate load was applied. Subjects were verbally encouraged to maintain maximal cadence throughout each 30-s sprint.

Measurements

Blood pressure and heart rate.

Before and following the intervention, as well as at the midintervention point, the BP at the brachial artery was measured using an automated sphygmomanometer (Dinamap Pro; GE Medical Systems, Tampa, FL). Following 10-min seated rest in an isolated room, three measurements were recorded. Mean arterial pressure (MAP) was calculated as 1/3 systolic pressure + 2/3 diastolic pressure. The means of the systolic pressure, diastolic pressure, and MAP measurements were used for data analysis.

Blood analysis.

Venous blood was sampled at rest (baseline) before each experimental test. Blood samples were also obtained at 1 min, at 3 min, and at task failure during the severe-intensity exercise bout. The blood samples collected during the severe-intensity exercise bout were drawn from a cannula (Insyte-WTM; Becton Dickinson, Madrid, Spain) inserted into the subject’s antecubital vein and were collected into lithium-heparin vacutainers (Becton Dickinson, Franklin Lakes, NJ). Blood [lactate] and [glucose], as well as plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] and [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$], were analyzed in all samples (square brackets denote concentration). Two hundred microliters of blood were immediately extracted from the lithium-heparin vacutainers and hemolyzed in 200 μl of Triton X-100 solution (Triton X-100; Amresco, Salon, OH) before blood [lactate] and [glucose] were measured (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). The remaining whole blood from each sample was centrifuged at 4,000 rpm for 8 min at 4°C within 2 min of collection. Plasma was immediately extracted, frozen at −80°C, and subsequently analyzed for [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] and [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] using chemiluminescence, as described by Wylie et al. (79).

Muscle biopsy.

Muscle samples were obtained from two incisions from the medial region of the vastus lateralis muscle under local anesthesia (1% lidocaine) using the percutaneous Bergström needle biopsy technique (7) with suction. Muscle samples were taken at three different time points before and following the intervention: at rest; following 3 min of severe-intensity exercise; and at task failure from severe-intensity exercise. The postexercise biopsies were taken while subjects remained on the cycle ergometer and were typically collected within 5–10 s of the completion of the exercise bout. Biopsy samples were immediately frozen in liquid nitrogen and stored at −80°C for subsequent analysis.

Muscle metabolites.

Following a freeze-drying process, samples were dissected to remove visible blood, fat, and connective tissue. Approximately 2 mg aliquots of isolated muscle fibers were weighed on fine-balance scales (Mettler Toledo XS105; Leicester, United Kingdom) and stored in 500-µl microcentrifuge tubes at −80°C. Prior to metabolite analysis, 200 µl of 3 M perchloric acid were added to ~2 mg dry wt muscle tissue. Following 3-min centrifugation and 30-min incubation on ice, 170 µl of supernatant were transferred to a fresh microcentrifuge tube, and 255 µl of cooled 2 M potassium bicarbonate (KHCO₃) were added. This was centrifuged, and the supernatant was analyzed for [PCr], [ATP], and [lactate] by fluorometric assays as previously described (51).

Muscle glycogen and pH.

Glycogen was extracted from ~1 mg dry wt muscle in 500 μl of 1 M hydrochloric acid (HCl) and hydrolyzed at 100°C for 2 h to glycosyl units, which were measured using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments) to determine muscle [glycogen]. Muscle pH was measured using a micro-pH meter (Sentron SI600; Roden, The Netherlands) following homogenization of ~1 mg dry wt muscle in a nonbuffering solution (145 mM KCl, 10 mM NaCl, and 5 mM NaF).

Muscle fiber type.

Approximately 20 mg of tissue obtained from each resting muscle biopsy sample were embedded in Tissue-Tek optimum cutting temperature compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands), rapidly frozen in liquid nitrogen-cooled isopentane, and stored at −80°C for subsequent histochemical analysis of myocellular characteristics. Serial cross sections (~10 μM thick) were cut in a cryostat (Cryostar NX50; Thermo Scientific) maintained at −16°C. Sections were mounted on three separate slides and preincubated at pH values of 4.3, 4.6, and 10.3. According to the lability to the acid and alkaline preincubation, the fibers were stained for myofibrillar ATPase, identified as type I, IIa, or IIx (9) and counted under an Olympus CKX41 microscope with cellSens Dimension software (Olympus, Tokyo, Japan). For each subject, 214 ± 104 fibers were analyzed, and each fiber type was expressed as a percentage of the total number counted.

Oxygen uptake.

The breath-by-breath V̇o₂ data from each step exercise test were initially examined to exclude values lying more than four SDs from the local mean. The filtered data were subsequently linearly interpolated to provide second-by-second values and time aligned to the start of exercise for each individual. The baseline V̇o₂ was defined as the mean V̇o₂ measured over the final 60 s of the 3-min baseline period. The end-exercise V̇o₂ was defined as the mean V̇o₂ measured over the final 60 s of exercise.

Statistical Analyses

Differences between groups in preintervention physiological and performance values were tested using a one-way ANOVA. Time-by-group ANOVAs with repeated measures for time were employed to determine the physiological and performance effects consequent to the interventions. In addition, one-way ANOVAs were used to assess differences between groups in the change values for physiological and performance variables preintervention to postintervention. All significant main and interaction effects were followed up by Fisher’s least significant difference post hoc tests. Data that were not normally distributed were log transformed before applying the ANOVA. All values are reported as means ± SD. Statistical significance was accepted at P < 0.05.

RESULTS

Compliance

All subjects within the training groups completed 100% of the training sessions and 100% of the sprints within each training session. All subjects reported that they fully adhered to the supplementation regimen and did not alter dietary and exercise behavior outside of their assigned group-specific intervention.

Plasma [$N{O}_3^{-}$] and [$N{O}_2^{-}$]

Preintervention resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] values were not different between groups (P > 0.05). A significant main effect for time (P < 0.001) and an interaction effect (P < 0.001) were observed for the plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] measured at rest. Compared with preintervention, SIT+BR increased resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] by ~590% at 2 wk and ~960% at 4 wk (both P < 0.001; Fig. 1A), and NT+BR increased resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] by ~505% at 2 wk and ~1,050% at 4 wk (both P < 0.001; Fig. 1A), but there was no change in resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] with SIT+PL (P > 0.05; Fig. 1A). Resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] was also greater at 4 wk compared with 2 wk in both SIT+BR and NT+BR (P < 0.05; Fig. 1A).

Fig. 1.

Mean ± SD resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] (A) and plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] (B) responses in SIT+BR (solid black line), SIT+PL (solid grey line), and NT+BR (dashed black line). *Different from preintervention (P < 0.05); †different from midintervention (P < 0.05); ‡different from SIT+PL (P < 0.05).

Preintervention resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] values were higher in SIT+PL (74 ± 62 nM) compared with SIT+BR (29 ± 19 nM; P < 0.05) and NT+BR (26 ± 13 nM; P < 0.05) but were similar between SIT+BR and NT+BR (P > 0.05). There was a significant main effect for time (P < 0.001) and an interaction effect (P < 0.001) for the plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] measured at rest. Compared with preintervention, SIT+BR increased resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] by ~485% at 2 wk and ~715% at 4 wk (both P < 0.001; Fig. 1B), and NT+BR increased resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] by ~600% at 2 wk and ~690% at 4 wk (both P < 0.001; Fig. 1B); however, there was no change in resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] with SIT+PL (P > 0.05; Fig. 1B). There were no differences in the plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] measured at rest between 2 and 4 wk in any of the groups (P > 0.05).

Blood Pressure

Systolic BP was not different between the groups before the interventions (P > 0.05; Table 1), but there was a significant main effect for time (P < 0.05) and an interaction effect (P < 0.05). Post hoc tests revealed that compared with preintervention, systolic BP was reduced at 2 and 4 wk (P < 0.05) by 5 ± 6 and 6 ± 4 mmHg, respectively, in SIT+BR (P < 0.05) and by 4 ± 5 and 10 ± 6 mmHg, respectively, in NT+BR (P < 0.05), whereas systolic BP remained unaltered in SIT+PL (P > 0.05; Table 1). Diastolic BP was not different between groups at preintervention (P > 0.05) and remained unaltered at 2 and 4 wk (both P > 0.05) in all interventions (Table 1). The MAP was not different between groups at preintervention but there was a significant main effect for time (P < 0.05) such that MAP was reduced by 3 ± 5 mmHg at 4 wk in both SIT+BR and NT+BR (P < 0.05) but was unchanged with SIT+PL (Table 1). Relative to postintervention resting baseline, plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] declined by ~65% at task failure during severe-intensity exercise (P < 0.001) in SIT+BR and NT+BR. The reduction in plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] following 3 min of severe-intensity exercise was greater in NT+BR compared with SIT+BR (P < 0.05).

Table 1.

Physiological and performance variables preintervention, midintervention, and postintervention

	SIT+PL			SIT+BR			NT+BR
	Pre	Mid	Post	Pre	Mid	Post	Pre	Mid	Post
Blood pressure
SBP, mmHg	116 ± 13	115 ± 14	116 ± 10	118 ± 11	113 ± 11*	112 ± 10*‡	117 ± 13	113 ± 10*	107 ± 17*‡
DBP, mmHg	67 ± 8	64 ± 7	66 ± 4	67 ± 9	64 ± 9	62 ± 7	63 ± 5	63 ± 7	62 ± 8
MAP, mmHg	83 ± 8	81 ± 8	83 ± 6	84 ± 8	80 ± 9	79 ± 7*	82 ± 7	80 ± 7	77 ± 7*
Incremental test
Peak WR, W	303 ± 78	306 ± 72	318 ± 73*†	298 ± 93	305 ± 90*	321 ± 91*†	296 ± 66	295 ± 67	300 ± 67*
Δ peak WR, W	—	4 ± 13	16 ± 15*†#	—	7 ± 10*#	24 ± 8*†#‡	—	0 ± 9	4 ± 4*
V̇o2peak, l/min	3.43 ± 0.99	3.49 ± 0.97	3.50 ± 0.86	3.19 ± 1.03	3.39 ± 1.06*	3.47 ± 1.02*	3.28 ± 1.03	3.42 ± 0.99	3.42 ± 1.08
V̇o2 at GET, l/min	1.55 ± 0.49	1.49 ± 0.41	1.62 ± 0.44	1.60 ± 0.37	1.58 ± 0.37	1.64 ± 0.43	1.61 ± 0.46	1.62 ± 0.52	1.61 ± 0.4
WR at GET, W	110 ± 32	103 ± 34	112 ± 27	102 ± 30	105 ± 32	110 ± 27*	105 ± 34	102 ± 29	112 ± 27
Moderate-intensity exercise
End-exercise V̇o2, l/min	1.57 ± 0.41	—	1.67 ± 0.44	1.64 ± 0.41	—	1.58 ± 0.42*‡	1.73 ± 0.32	—	1.65 ± 0.34*‡
Severe-intensity exercise
Time to task failure, s	297 ± 69	—	460 ± 186*#	248 ± 53	—	418 ± 132*#	266 ± 82	—	275 ± 84

Values are means ± SD. SIT+PL, high-intensity interval training plus $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-depleted beetroot juice; SIT+BR, high-intensity interval training plus $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice; NT+BR, no-training plus $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich beetroot juice. Pre, preintervention; mid, midintervention; post, postintervention; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; WR, work rate; V̇o₂, oxygen uptake; V̇o_2peak, peak V̇o₂; GET, gas exchange threshold.

^*Different from preintervention (P < 0.05);

^†different from midintervention (P < 0.05);

^#different from NT+BR (P < 0.05);

^‡different from SIT+PL (P < 0.05).

Incremental Exercise Test

Peak WR was not different between the groups at preintervention (P > 0.05; Table 1). There was a significant main effect by time (P < 0.001) and an interaction effect (P < 0.05). Post hoc tests revealed that peak WR was improved at 4 wk compared with preintervention in all groups (P < 0.05; Table 1). However, peak WR increased more from preintervention to postintervention in SIT+BR than in SIT+PL (P < 0.001; Fig. 2). Additionally, peak WR was improved at 2 wk compared with preintervention in SIT+BR only (P < 0.05; Fig. 2).

Fig. 2.

Mean ± SD changes (Δ) in peak WR at midintervention and postintervention in the three groups expressed relative to preintervention baseline. The change in peak WR from preintervention to postintervention was greater in SIT+BR (solid black line) than in SIT+PL (solid grey line) and NT+BR (dashed black line). *Different from preintervention (P < 0.05); †different from midintervention (P < 0.05); #different from NT+BR (P < 0.05); ‡different from SIT+PL (P < 0.05).

V̇o_2peak was not different between the groups at preintervention (P > 0.05; Table 1). There was a significant main effect by time on V̇o_2peak (P < 0.05). Post hoc analysis revealed that compared with preintervention, V̇o_2peak was increased after 2 and 4 wk with SIT+BR (P < 0.05; Table 1) but remained unchanged in SIT+PL and NT+BR (P > 0.05; Table 1). However, there were no differences between the three groups in the change in V̇o_2peak from preintervention to postintervention (P > 0.05). There were no significant changes in body mass from preintervention to postintervention in any of the groups.

The V̇o₂ at the GET was not different between the groups at preintervention (P > 0.05; Table 1) and was not altered by any intervention (P > 0.05; Table 1). The WR associated with the GET was not different between groups at preintervention (P > 0.05; Table 1). There was a significant main effect for time such that the WR at the GET was increased preintervention to postintervention in SIT+BR only (P < 0.05; Table 1). However, there were no differences between the three groups in the change in the WR at the GET from preintervention to postintervention (P > 0.05).

Step Exercise Tests: Moderate-Intensity Exercise

The V̇o₂ measured during baseline cycling at 20 W preceding the transition to moderate-intensity exercise was not different between groups at preintervention (P > 0.05; Table 1) and was not affected by any intervention (P > 0.05; Table 1). The end-exercise V̇o₂ during moderate-intensity exercise was not different between groups at preintervention (P > 0.05; Table 1). There was a significant main effect by time (P < 0.05) and an interaction effect (P < 0.05) on end-exercise V̇o₂. Post hoc analyses revealed that compared with preintervention, end-exercise V̇o₂ was significantly reduced in SIT+BR (P < 0.05) and NT+BR (P < 0.05) but was unaltered in SIT+PL (P > 0.05; Table 1). There was no difference in the change in end-exercise V̇o₂ from preintervention to postintervention between the SIT+BR and NT+BR groups (P > 0.05).

Step Exercise Tests: Severe-Intensity Exercise

The time to task failure during severe-intensity exercise was not different between groups at preintervention (P > 0.05; Table 1). There was a significant main effect by time (P < 0.05) and an interaction effect (P < 0.05) such that time to task failure was improved by 163 ± 144 s preintervention to postintervention in SIT+PL (P < 0.05; Table 1) and by 170 ± 90 s preintervention to postintervention in SIT+BR (P < 0.05; Table 1) but was unaltered by NT+BR (P > 0.05; Table 1). There was no difference in the change in the time to task failure from preintervention to postintervention between the SIT+BR and SIT+PL groups (P > 0.05).

Blood [lactate] was not different between groups during severe-intensity exercise at preintervention (P > 0.05). There was a main effect by time on blood [lactate] (P < 0.05). Post hoc analysis revealed that blood [lactate] was lower at 1 min (1.2 ± 1.1 mM decrease from same time point preintervention; P < 0.05; Fig. 3) and at 3 min (1.6 ± 1.5 mM decrease from same time point preintervention; P < 0.05, Fig. 3) during severe-intensity exercise in SIT+BR but not SIT+PL or NT+BR (P > 0.05). Further analyses revealed that the increase in blood [lactate] from rest to 3 min was attenuated postintervention compared with preintervention in SIT+BR (2.7 ± 0.9 vs. 3.9 ± 0.8 mM; P < 0.05). This attenuation was significantly greater than the equivalent change in blood [lactate] from rest to 3 min in SIT+PL (postintervention 4.1 ± 1.9 vs. preintervention 3.7 ± 1.2 mM; P < 0.05).

Fig. 3.

Mean ± SD blood [lactate] at rest (black bars), at 1 min (patterned bars), at 3 min (grey bars), and at task failure (open bars) during severe-intensity exercise. *Different from preintervention (P < 0.05).

Muscle Substrates and Metabolites

Preintervention values for muscle substrates and metabolites during severe-intensity exercise were not different between groups (P > 0.05), and muscle [ATP] and [PCr] were unchanged by the interventions in all groups (P > 0.05).

There were main effects by time on the muscle [lactate] and pH measured at 3 min of severe-intensity exercise (P < 0.05). Post hoc tests revealed that compared with preintervention, muscle [lactate] was lower and pH was higher at 3 min of severe-intensity exercise postintervention in SIT+BR (P < 0.05; Fig. 4). Further analyses revealed that compared with preintervention, the increase in muscle [lactate] and the decrease in muscle pH from rest to 3 min of exercise tended to be attenuated postintervention in SIT+BR (both P = 0.09).

Fig. 4.

Mean ± SD muscle [lactate] (A), muscle pH (B), and muscle [glycogen] (C) at rest (black bars), at 3 min (grey bars), and at task failure (open bars) during severe-intensity exercise. d.w., Dry weight. *Different from preintervention (P < 0.05); #different from postintervention NT+BR (P < 0.05); ‡different from postintervention SIT+PL (P < 0.05).

There was a main effect by time on muscle [glycogen] measured at rest, at 3 min, and at task failure (all P < 0.05). Post hoc tests revealed that muscle [glycogen] was higher at all three time points postintervention compared with preintervention in SIT+BR (P < 0.05; Fig. 4C). Muscle [glycogen] was also higher at all three time points postintervention in SIT+BR and SIT+PL compared with NT+BR (P < 0.05; Fig. 4). There were no differences between SIT+BR and SIT+PL in the change in muscle [glycogen] from preintervention to postintervention at rest, at 3 min of exercise, or at task failure (P > 0.05).

Muscle Fiber Type

The relative proportion of type I (SIT+BR, 57 ± 16%; SIT+PL, 59 ± 10%; NT+BR, 48 ± 16%), type IIa (SIT+BR, 36 ± 12%; SIT+PL, 36 ± 16%; NT+BR, 44 ± 16%), and type IIx (SIT+BR, 7 ± 8%; SIT+PL, 5 ± 7%; NT+BR, 8 ± 12%) muscle fibers at preintervention was not different between groups (P > 0.05). There was a significant effect of time and an interaction effect on the proportion of type IIx fibers. Post hoc tests revealed that the proportion of type IIx fibers identified in SIT+BR was lower postintervention (4 ± 5%) compared with preintervention (7 ± 8%; P < 0.05). In contrast, the proportion of type IIx fibers identified in SIT+PL tended to be higher postintervention (10 ± 9%) compared with preintervention (5 ± 7%; P = 0.07). The change in type IIx fibers was significantly different in SIT+BR compared with SIT+PL (P < 0.05) but not NT+BR (P > 0.05). There were no differences in the proportion of type I (SIT+BR, 55 ± 12%; SIT+PL, 58 ± 10%; NT+BR, 50 ± 17%) or type IIa (SIT+BR, 41 ± 9%; SIT+PL, 32 ± 16%; NT+BR, 43 ± 14%) muscle fibers following any intervention (P > 0.05). However, there was a significant interaction effect on the proportion of type I and type IIa fibers combined (type I+IIa; P < 0.05). Post hoc tests revealed that the proportion of type I+IIa fibers identified in SIT+BR was higher postintervention (96 ± 6%) compared with preintervention (93 ± 8%; P < 0.05). In contrast, the proportion of type I+IIa fibers identified in SIT+PL tended to be lower postintervention (90 ± 9%) compared with preintervention (95 ± 7%; P = 0.07). The change in type I+IIa fibers was significantly different in SIT+BR and NT+BR compared with SIT+PL (P < 0.05).

DISCUSSION

This is the first study to investigate the combined effect of SIT and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation, administered in the form of beetroot juice, on muscle metabolic adaptations and the physiological responses to ramp incremental, moderate-intensity and severe-intensity exercise performance in normoxia. We compared the effects of chronic $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation alone (NT+BR) with the effects of concurrent $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich (SIT+BR) and $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-depleted (SIT+PL) beetroot juice supplementation during a SIT intervention. Consistent with our hypotheses, the separate 4-wk interventions of SIT and chronic BR supplementation independently induced several beneficial physiological and/or performance effects. However, the main finding of the present study was that the combination of SIT and BR supplementation provided greater improvements in incremental exercise performance compared with either intervention alone and led to greater improvements in some indexes of muscle metabolic adaptation.

Plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] and [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] were elevated, and systolic BP was lowered following 2 and 4 wk of BR supplementation, changes which are consistent with elevated systemic NO bioavailability. Interestingly, however, resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] and [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] were not altered following 4 wk of SIT+PL. Previous studies have reported that subjects with higher aerobic fitness and/or training status have higher resting plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$] and [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] compared with less fit and/or sedentary subjects (53, 63). The results of the present study may therefore indicate that short-term SIT, at least when combined with PL supplementation, does not substantially modify nitric oxide synthase activity or protein expression. The reduction in plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] from resting baseline to task failure during severe-intensity exercise was similar between NT+BR and SIT+BR (~65% decline). However, the reduction in plasma [$\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$] from resting baseline to 3 min of severe-intensity exercise was attenuated in SIT+BR (~25% decline) compared with NT+BR (~45% decline). It is possible that this may be related to differences in training status induced by the separate interventions; for example, less reduction of $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ to NO may have been required following SIT+BR because of training-related improvements in muscle capillarity and oxygenation (18).

The reductions in systolic BP (SIT+BR, −4 and −5%; NT+BR, −6 and −9%; at 2 and 4 wk, respectively) reported in the present study are similar to those previously reported in healthy volunteers following shorter supplementation periods (5, 44, 73, 74). Diastolic BP was unaltered in NT+BR and SIT+PL but was reduced by 7% in SIT+BR. MAP was lowered by ~4% in both $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-supplemented groups (SIT+BR and NT+BR) but was unaltered in SIT+PL. Collectively, these data indicate that 4 wk of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation may result in a greater reduction in BP than 4 wk of SIT alone.

Effect of SIT and BR on Submaximal V̇o₂ and V̇o_2peak

A high exercise economy, i.e., a low V̇o₂ for a given power output, is an important determinant of exercise performance (33). It has been postulated that exercise training can lower the O₂ cost of submaximal cycling (55). However, in the present study, the O₂ cost of moderate-intensity exercise was only reduced following training in SIT+BR, and the magnitude of the reduction in the O₂ cost of exercise was not different from that observed with NT+BR, suggesting that 4 wk of SIT per se has no influence on the O₂ cost of submaximal cycling. This finding is consistent with previous work indicating that the O₂ cost of exercise may be reduced by dietary $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation (5, 45, 46, 73). The physiological bases for the improved efficiency following $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ ingestion are likely related to a reduced ATP cost of muscle force production (6) and/or a reduced O₂ cost of mitochondrial ATP resynthesis (46, cf. 76).

Despite the low training volume, SIT has emerged as a potent strategy to increase aerobic capacity and endurance exercise performance in as little as 2 wk (11, 65, 70). We found that V̇o_2peak was not significantly altered by 4 wk of either NT+BR or SIT+PL. The former result is consistent with the majority of studies that have assessed V̇o_2peak following acute or short-term $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation (5, 6, 39, 45, 78)_. The lack of effect of SIT on V̇o_2peak is also consistent with some (11–13, 26), but not all (65, 70), previous investigations. The physiological and muscle metabolic adaptations to SIT are likely dependent upon the initial training status of the subjects along with the exact nature of the training stimulus, including the frequency and duration of both the sprint and recovery periods (66). In this respect, it is important to highlight that our exercise-training protocol was shorter in duration compared with some studies (13) and the progression in training volume was more gradual than in other studies (4, 65) in which V̇o_2peak was increased.

Although the change in V̇o_2peak from preintervention to postintervention was not different between the three groups, the increase in V̇o_2peak was only greater from preintervention to postintervention in SIT+BR, suggesting that $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation may enhance the adaptation of V̇o_2peak to SIT. Further work is required to confirm this observation and to elucidate the potential cardiovascular and/or metabolic mechanisms which may be responsible.

Effect of SIT and BR on Exercise Performance

The peak WR during incremental exercise at 4 wk was improved in both training groups. It is notable that peak WR was also significantly improved following NT+BR. Although this effect was small, it is consistent with an earlier study which reported a significant increase in peak WR during incremental exercise following 15 days of BR supplementation (73). A greater peak WR at 2 wk of training was only observed with SIT+BR. Moreover, the improvement in peak WR at 4 wk was greater in SIT+BR than in SIT+PL and NT+BR. The greater, and more rapidly attained, improvements in incremental exercise test performance with SIT+BR is presumably a function of the improved exercise economy and/or favorable muscle metabolic profile which would be expected to result in an extended time to reach V̇o_2peak. Our results are consistent with a recent study by Muggeridge et al. (57) which reported that 3 wk of SIT (4–6 repeated 15-s sprints) increased peak WR during incremental exercise to a greater extent when subjects were supplemented with $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ compared with placebo.

The time to task failure during severe-intensity exercise was significantly increased after 4 wk of both SIT+BR (group mean change +69%) and SIT+PL (+55%), but not NT+BR (+3%). Despite evidence for an enhanced muscle metabolic response to severe-intensity exercise in SIT+BR compared with SIT+PL (see below), this did not translate into a greater improvement in severe-intensity exercise performance. It is not clear why ramp incremental exercise test performance was improved with SIT+BR when time to task failure during severe-intensity exercise was not, although greater variability in time-to-exhaustion tests may have contributed to the difference (20). Indeed, it is interesting to note that the improvement in time to task failure ranged from 37 to 116% in SIT+BR (with 9/12 subjects improving by >50%) and from 4 to 122% in SIT+PL (with 4/12 subjects improving by >50%). Our results are similar to those of Puype et al. (64), who found that 6 wk of endurance training in normobaric hypoxia with BR supplementation did not improve 30-min time trial performance relative to the placebo condition. However, it remains unclear whether BR supplementation during training could improve performance in other types of exercise. Recent studies indicate that BR may be ergogenic during high-intensity intermittent exercise (2, 71, 78) and that compared with placebo, $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation during SIT improves fatigue resistance during repeated sprint exercise (57) and may enhance mean power output in a 30-s sprint (21). Further studies are required to investigate whether the subtle enhancements of skeletal muscle adaptation to training with BR might translate into improved performance during these other forms of exercise.

Effect of SIT and BR on the Muscle Metabolic Response to Exercise

Although conflicting data exist, SIT has been implicated in rapid skeletal muscle remodeling (11–13, 26). The extreme perturbations in substrate availability and metabolite accumulation caused by repeated sprint efforts require substantial oxidative energy turnover to restore homeostasis (8). The fluctuations in ATP availability and local O₂ tension are potent stimulators of signaling pathways and may induce mitochondrial biogenesis and oxidative enzyme adaptation via the transcription of PGC-1α (28, 31, 47). Recent findings indicate that dietary $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ may favorably affect the contractility (30, 32) and perfusion (22, 23) of type II muscle fibers and reduce the energetic cost of muscle force production during high-intensity exercise (6, 24). Similar to SIT (3, 13, 48–50, 61, 62), elevating $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ and NO bioavailability with chronic $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$-rich BR supplementation may also stimulate the transcription of PGC-1α (43, 54, 58), a key regulator of mitochondrial biogenesis (77) and angiogenesis (1, 15). We therefore determined the effects of 4-wk SIT and 4-wk BR supplementation on the muscle metabolic responses during exercise and tested the hypothesis that these adaptations may be amplified when the interventions were combined.

There were no differences in muscle [ATP], [PCr], [lactate], or pH at rest or at task failure during severe-intensity exercise, postintervention compared with preintervention, in any group. However, at 3 min into severe-intensity exercise, there was evidence of reduced metabolic perturbation, postintervention compared with preintervention, in the SIT+BR group only. Specifically, muscle [lactate] as well as blood [lactate] was lower, and muscle pH was higher, at 3 min of severe-intensity exercise following SIT+BR but not SIT+PL or NT+BR (Figs. 3 and 4), suggesting an enhanced muscle metabolic adaptation to SIT when combined with BR supplementation.

The reason for the small difference in muscle acidosis at the 3-min exercise isotime with SIT+BR compared with SIT+PL is unclear. However, this may be the result of differences in exercise efficiency between the training groups. The lower O₂ cost of exercise measured at the same submaximal work rate in SIT+BR would be expected to lower the physiological strain and potentially reduce substrate-level phosphorylation and lactate production during exercise (34). Furthermore, BR supplementation has been shown to elevate microvascular Po₂ in type II muscles of exercising rats thus promoting O₂ exchange between the capillary and the myocyte and enabling a better preservation of intramuscular homeostasis (22, 23). By better maintaining oxidative function, this mechanism may be important in delaying lactate accumulation during severe-intensity exercise, which is known to mandate an increased recruitment of type II fibers to sustain power output (42). While $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ intake alone would be expected to promote some of these effects (e.g., a lower O₂ cost of submaximal exercise in the NT+BR group in the present study), $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ intake combined with training may synergistically improve the muscle metabolic response to severe-intensity exercise. In particular, the SIT+BR group evidenced improved exercise efficiency (which was observed with NT+BR but not SIT+PL) and improved performance and physiological responses/adaptations to maximal exercise (which were observed with SIT+PL but to a much lesser extent with NT+BR).

None of the interventions influenced the proportion of type I muscle fibers identified following training. Interestingly, there was a disparity in the muscle phenotypic response to training between SIT+BR and SIT+PL. Specifically, SIT+BR resulted in a significant reduction in the proportion of type IIx muscle fibers. In contrast, SIT+PL resulted in a trend toward a greater proportion of type IIx fibers following the intervention period. These results suggest that a remodeling of skeletal muscle toward a more oxidative phenotype following SIT (26, 27) may be facilitated by BR supplementation and perhaps hampered by PL supplementation. Our findings are consistent with a recent study which also reported changes in muscle fiber type composition following 5 wk of SIT with ~5-mmol daily $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation (21). These authors reported that SIT performed in hypoxia resulted in a significant increase in the relative number of type IIa fibers in the vastus lateralis muscle (from ~45 to 56%) when subjects ingested $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ compared with placebo. It is possible that the differences in the muscle metabolic or performance response to exercise following SIT when combined with $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ compared with placebo supplementation (present study; 57) are related to changes in muscle fiber type composition, i.e., a greater reduction in type IIx fibers and/or a greater increase in type IIa fibers (present study; 21).

It is important to highlight that both the BR and PL supplements contain high concentrations of antioxidants including betacyacins and polyphenols (38, 67) which may potentially interfere with skeletal muscle adaptations to training (56, 60). It is possible, therefore, that the adaptations to training in the SIT+PL group were attenuated in the present study because of the simultaneous intake of antioxidants. However, it is also possible that the potential for chronic $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ administration to enhance muscular adaptations and exercise performance with SIT was underestimated in the SIT+BR group for the same reason. On the other hand, it has recently been reported that BR supplementation increases hydrogen peroxide emission from the mitochondria, an effect that could promote redox signaling (76) and enhance training adaptations. Moreover, the combination of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ with antioxidants might promote the reduction of $\mathrm{N}{\mathrm{O}}_{\mathrm{2}}^{\mathrm{-}}$ to NO and facilitate physiological effects (36). Further research should investigate the influence of $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ alone (as NaNO₃ or KNO₃) and BR on the skeletal muscle adaptations to training. Our study design involved 4 wk of daily BR supplementation with the final dose being consumed on the morning of the postintervention laboratory tests. Our measurements therefore reflect the combined effects of chronic and acute BR (or PL) supplementation superimposed on exercise training. It has been reported recently that 4 wk of BR supplementation continue to exert physiological effects for at least 48 h following the cessation of supplementation (80). Future studies might therefore be designed to partition out the influence of chronic $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ or BR supplementation (without additional acute supplementation) on the adaptations to training.

Conclusions

In the absence of training, chronic BR ingestion resulted in a significant reduction in the O₂ cost of moderate-intensity exercise and a small but significant increase in peak WR. SIT+PL resulted in improvements in peak WR during incremental exercise and time to task failure during severe-intensity exercise. Greater changes in peak WR during incremental exercise were found with SIT+BR compared with SIT+PL and NT+BR. In addition, type IIx muscle fiber proportion was reduced, and at the 3-min isotime during severe-intensity exercise, muscle pH was higher and muscle (and blood) [lactate] was lower in SIT+BR only. These findings suggest that the independent physiological and performance effects of SIT and BR supplementation may be enhanced when these interventions are combined. Dietary $\mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ supplementation in the form of BR may potentiate some exercise performance and muscle metabolic adaptations to SIT.

GRANTS

This study was supported by PepsiCo, Illinois, Grant 2012–1891470. J. Fulford’s salary was supported via a National Institute for Health Research grant.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

C.T., L.J.W., J.R.B., J.F., M.I.B., J.K., S.T.M., S.J.B., A.V., and A.M.J. performed experiments; C.T., L.J.W., J.R.B., J.F., A.V., and A.M.J. analyzed data; C.T., L.J.W., J.F., J.C., S.J.B., A.V., and A.M.J. interpreted results of experiments; C.T., L.J.W., and A.M.J. prepared figures; C.T., L.J.W., J.C., S.J.B., A.V., and A.M.J. drafted manuscript; C.T., L.J.W., J.R.B., J.F., M.I.B., J.K., S.T.M., J.C., S.J.B., A.V., and A.M.J. edited and revised manuscript; C.T., L.J.W., J.R.B., J.F., M.I.B., J.K., S.T.M., J.C., S.J.B., A.V., and A.M.J. approved final version of manuscript.