Nitric oxide (NO) is an important signaling molecule within the body, generated endogenously by the oxidation of L-arginine (1). NO can also be generated through the reduction of dietary inorganic nitrate to nitrite, by facultative bacteria in the oral cavity, with nitrite further reduced to NO within various tissues around the body (2). It is through this nitrate–nitrite pathway that supplementing the diet with inorganic nitrate seems to increase the bioavailability of NO and have measurable physiological effects, including reduced blood pressure (3,4), increased exercise economy (4–6), and improved endurance performance (5,7). These effects have been widely investigated over the last decade (8), but only recently has evidence suggested that nitrate supplementation may also enhance the excitation–contraction coupling of skeletal muscle, resulting in greater force production for a given excitation (9–11).
Hernandez et al. (10) supplemented the diet of rats with nitrate for 7 d and reported improved tetanic peak force at low (≤50 Hz) stimulation frequencies in fast- but not slow-twitch muscle fibers, which was associated with increased release of Ca2+ from the sarcoplasmic reticulum (SR). Three in vivo human studies have since investigated involuntary contractile responses of the mixed-fiber quadriceps muscles after dietary supplementation with nitrate-rich beetroot juice, and although two reported 5%–20% improvements in low-frequency (≤20 Hz) tetanic peak force (9,11), the third reported no effects (12). These studies used different control conditions—nitrate-depleted beetroot juice (12), lime cordial (9), and no placebo (11)—to compare with the nitrate supplement condition, which may have contributed to the inconsistent results. Furthermore, the benefits of nitrate supplementation to contractile performance seem specific to fast-twitch fibers (10), and so will likely be diluted and thus variable in human whole mixed-fiber muscles. Nevertheless, assuming that it is possible to improve excitation–contraction coupling and enhance low-frequency force in human muscle, this would theoretically benefit humans during voluntary contractions, where free cytosolic Ca2+ is low or rising, such as during repeated submaximal voluntary contractions or during the rising slope of the force–time curve of explosive voluntary contractions. The latter was investigated by Haider and Folland (9), and although nitrate supplementation improved explosive force during involuntary twitch and 300-Hz tetanic contractions, explosive force during voluntary contractions was unaffected. This poor translation of effects from involuntary to voluntary contractions may have been due to the large variability in neural drive during voluntary explosive contractions (13), which is a more important determinant of explosive voluntary force than involuntary explosive force (13,14). It is conceivable, however, that the influence of nitrate supplementation on explosive voluntary force may become more apparent in fatigued conditions where excitation–contraction coupling is disrupted.
A common feature of neuromuscular fatigue is the greater reduction of force at low (<50 Hz) versus high (≥50 Hz) stimulation frequencies (low-frequency fatigue ), thought to be largely caused by reduced SR Ca2+ release (16–18), and reflective of disruption to excitation–contraction coupling. Low-frequency fatigue seems to have two components: one dependent on metabolite accumulation, observed immediately after fatiguing exercise of sufficiently high force–time integral (19,20) but which recovers within minutes (16,19), and one observed after several minutes of recovery which can last for hours (15,19) and is thus independent of metabolite accumulation. Recent evidence of nitrate supplementation increasing SR Ca2+ release in unfatigued rat muscle (10) and reducing metabolic perturbation during fatiguing contractions in humans (21) raises the possibility that nitrate supplementation may attenuate the first component of low-frequency fatigue by countering the mechanisms causing it. Hoon et al. (12) reported a reduction in the fatigue of low-frequency (20 Hz) peak force during repeated contractions with hypovolemia, despite observing no effects in unfatigued conditions, suggesting that the influence of nitrate supplementation becomes more evident during fatiguing conditions known to disrupt excitation–contraction coupling. However, Hoon et al. (12) did not quantify low-frequency fatigue (low- vs high-frequency forces), so the extent of disruption to excitation–contraction coupling in their protocol and the benefit of nitrate supplementation to any disruption remain unclear. Moreover, it is unknown whether the effects of nitrate supplementation on explosive voluntary force also become more evident with fatiguing exercise that disrupts excitation–contraction coupling.
The purpose of this study was to investigate the effects of dietary nitrate supplementation on involuntary and voluntary contractile responses in human muscle during both unfatigued and fatigued conditions. We hypothesized that in unfatigued conditions, nitrate supplementation would enhance low-frequency force but not affect explosive voluntary force, whereas in fatigued conditions, nitrate supplementation would attenuate both low-frequency fatigue and the loss of explosive voluntary force. The fatiguing protocol used in this study was a 5-min all-out bout of 60 maximal voluntary contractions (MVC) in which mean MVC force declines to a plateau representing a critical force threshold (22) due to the depletion of high-energy phosphates and considerable metabolite accumulation (23). This protocol was chosen to enable between-condition differences in force–time characteristics of MVC during the protocol, while simultaneously ensuring a plateau in fatigue and metabolic perturbation before testing the involuntary contractile responses immediately after the protocol. We also predicted that the protocol would provide sufficient metabolic stress and force–time integral to observe the first component of low-frequency fatigue.
Seventeen healthy, nonsmoking, recreationally active men (mean ± SD age, 23 ± 4 yr; body mass, 74.04 ± 9.62 kg; height, 1.75 ± 0.06 m) volunteered to participate in this study, which was approved by the University of Roehampton Ethical Advisory Committee. Participants provided written informed consent before their involvement.
Similar to the design of Haider and Folland (9), each participant visited the laboratory at a consistent time of day on four separate occasions to complete two familiarization and two experimental trials. Seven days separated each of the first three trials, whereas the two experimental trials were separated by 9 d and completed in a randomized, double-blinded order. In the 7 d immediately before each experimental trial, participants supplemented their diet with either nitrate-rich (NIT) or nitrate-depleted (PLA) beetroot juice. During the course of the study, participants were requested to maintain habitual physical activity and diet, not use antibacterial mouthwash, and abstain from caffeine (for 6 h), alcohol (for 24 h), and vigorous exercise (for 36 h) before experimental trials.
Each trial involved the same protocol of isometric voluntary and involuntary contractions of the knee extensors of the dominant leg, determined as the preferred leg to kick a ball with. In the protocol, participants first completed explosive MVC and electrically evoked 1-s tetanic contractions at 10, 20, 50, and 100 Hz, to determine neuromuscular function in unfatigued conditions. Participants then completed a fatiguing protocol of 60 MVC, followed immediately by the same series of tetanic contractions as that mentioned previously to determine neuromuscular function in fatigued conditions. External knee extensor force and surface EMG were recorded throughout the measurement trials. Finger-prick blood samples were collected at the start of each experimental trial to determine plasma nitrate and nitrate concentrations. Data analysis was completed before un-blinding the investigators to the condition order for each participant.
Participants supplemented their diet with 70-mL shots of concentrated NIT (400–500 mg per 70 mL) or PLA (0.35–1.26 mg per 70 mL) beetroot juice (nonorganic SPORT shot, Beet It; James White Drinks Ltd, Ipswich, UK). Two shots were taken per day for the 7-d supplementation period, one each morning and one each evening, except for the day of the experimental trial when both shots were taken together 2.5 h before the trial. Daily nitrate supplementation was ~12.9 mmol and ~0.01–0.04 mmol in the NIT and PLA conditions, respectively.
Force and EMG Recordings
Participants sat in a custom-built, low-compliance, isometric knee extensor strength testing chair (9), with hip and knee angles of 100° and 120°, respectively (180° = anatomical position). Shoulder and pelvis strapping secured participants tightly to the chair, minimizing upper body movements. An ankle strap (35-mm width reinforced canvas webbing) was placed around the leg being tested, at a constant 4 cm above the lateral malleolus. The strapping was in series with a linear-response S-beam strain gage load cell (1.5-kN maximum amplitude and amplitude resolution of 1/2000; Force Logic, Swallowfield, UK), positioned perpendicular and posterior to the shank. The force signal was amplified (×370), sampled at 2000 Hz via an AD convertor (Micro 1401; CED, Cambridge, UK) and recorded on a PC using Spike2 software (CED). Offline, the force signal was low-pass filtered at 500 Hz with a fourth-order zero-lag Butterworth filter and corrected for the weight of the shank by subtracting resting baseline force.
After preparation of the skin (shaving, lightly abrading, and cleansing with 70% ethanol), single differential surface EMG electrodes (2-cm diameter Ag–Ag–Cl gel; 2-cm interelectrode distance; Noraxon USA, Inc, Scottsdale, AZ) were placed over the belly of the muscle of the rectus femoris, vastus lateralis, and vastus medialis. Electrodes were positioned parallel with the presumed orientation of the fibers, and at ~50% (rectus femoris), ~54% (vastus lateralis), and ~88% (vastus medialis) of the distance between the greater trochanter and the lateral femoral condyle. EMG signals were filtered (10 Hz, high pass) and amplified (×200) at the source (TeleMyo DTS; Noraxon USA, Inc), transmitted wirelessly to the DTS desktop receiver for further amplification (total system gain ×500), and sampled at 2000 Hz via the same AD convertor and PC software as the force signal. Offline, EMG signals were corrected for the 156-ms delay inherent in the Noraxon wireless system and band-pass filtered between 10 and 500 Hz with a fourth-order zero-lag Butterworth filter. All measurements of EMG amplitude (see Fatigue Protocol section) were averaged across the three quadriceps muscles to give a single mean value for the knee extensors.
Unfatigued Voluntary Contractions
After a series of warm-up contractions (two, 3-s contractions each at 30%, 50%, 70%, and 90% of perceived maximal effort), participants completed 10 MVC (each separated by ~60 s) in which they were instructed to push as “fast and hard” as possible for ~3 s from a relaxed (zero active tension) state and without prior countermovement. Participants were instructed to focus on pushing fast in the early phase (first second) of the MVC, followed by as hard as possible after that to maximize explosive and maximal forces, respectively (24). Biofeedback was provided on a computer monitor in front of participants, displaying (i) the force signal with a cursor on the greatest force achieved so far that session, (ii) the resting baseline force on a sensitive scale to provide feedback on whether a countermovement or pretension had occurred before the MVC, and (iii) the slope of the force–time curve (40-ms time constant) with a cursor on the highest peak slope achieved so far that session. Verbal encouragement was provided throughout.
All data were analyzed using custom-developed computer programs in Matlab (The MathWorks Inc, Natick, MA). Maximal voluntary force (MVF) was determined as the greatest peak force recorded in any MVC performed in that session. Explosive impulse (force–time integral) was measured over the first 50 (IMP0–50), 100 (IMP0–100), and 150 ms (IMP0–150) from force onset and was averaged across the three valid MVC with the highest IMP0–100, in the unfatigued condition. Force onset was defined as the last data point before the slope of the force–time curve (2-ms time constant) crossed and remained above zero for the time it took force to reach 50% MVF. Valid MVC for explosive impulse measures were considered those that had no pretension or countermovement before force onset, determined via the following criteria: (i) mean baseline force in the 200-ms immediately before force onset was between −1% and 1% MVF, and (ii) force at onset was within 1 N of this mean baseline force.
Unfatigued Tetanic Contractions
One-second, tetanic contractions were evoked with a train of square-wave electrical impulses (0.2-ms pulse width; DS7AH, Digitimer Ltd, UK) via two carbon rubber electrodes (14 × 10 cm; Electro Medical Supplies, Wantage, UK) placed ~8 cm apart at proximal (anode) and distal (cathode) ends of the anterior surface of the thigh. Starting at a near imperceptible electrical current (~20 mA), 100-Hz tetanic contractions were evoked at 20-s intervals, and the current intensity was gradually increased (20–30 mA steps) with each contraction until the peak force response reached 50% of the MVF measured in the familiarization session (typically at 100–150 mA). At this stimulation intensity, two sets of four tetanic contractions were evoked with one contraction each at 10, 20, 50, and 100 Hz per set. The order of the four different stimulation frequencies within a set was randomized between participants, but remained constant for both sets and conditions (i.e., NIT and PLA) for each participant. Two seconds separated consecutive tetanic contractions within and between sets.
Tetanic peak force was determined as peak instantaneous force for 10-Hz contractions, or mean force calculated over a 300-ms period at peak instantaneous force (150 ms either side of peak, or 300 ms before peak if at the end of the plateau) for 20-, 50-, and 100-Hz contractions. Tetanic peak rate of force development (RFD) was determined as the peak instantaneous slope of the force–time curve over a 25-ms moving time window. For each frequency, peak forces and RFD were averaged across the two sets, and calculated relative to peak force or RFD at 100 Hz, respectively, controlling for any differences in stimulation intensity between conditions. The 20/50-Hz ratio was also measured for both peak force and RFD to assess any differential effects of condition on low (20 Hz) versus high (50 Hz) frequencies.
After a 10-min recovery out of the strength testing chair, participants repeated the warm-up outlined earlier before completing a fatigue protocol involving 60, 3-s MVC (each separated by 2 s). This fatigue protocol has previously been shown to elicit a decline in mean force with each MVC to an asymptotic plateau (critical force threshold) by the last 6 MVC, reflecting the highest force that can be maintained with a metabolic steady state (22,23). The instruction with each MVC was as previously mentioned for the unfatigued MVC (i.e., “push fast and hard”), but participants were also instructed to relax as quickly as possible at the end of each MVC in preparation for the next MVC in the series. The timing of each MVC was maintained using a digital metronome (Tempo Application for IPad; FrozenApe.com). Participants were instructed not to pace themselves, but to produce a maximal effort with each MVC, and were blinded from the time and MVC number until immediately before the last MVC. Two horizontal cursors were placed at 90% and 85% of MVF recorded in the unfatigued MVC, and participants were required to exceed these forces in the first and second MVC of the protocol, respectively. Failure to do so was considered indicative of submaximal efforts from the start, in which case the protocol was interrupted, 5-min recovery given, and the protocol reattempted, for a maximum of three attempts. The same two sets of tetanic contractions as performed in unfatigued conditions were completed, commencing at a similar 3.0 ± 1.1 s (PLA) and 2.6 ± 0.8 s (NIT; paired comparison, P = 0.278) after the last MVC in the fatigue protocol.
For each MVC in the fatigue protocol, the force–time integral (impulse), mean force, and root mean squared (RMS) EMG amplitude were calculated between the data points where force increased above and decreased below 2% MVF. This threshold for detecting MVC onset and offset was selected for computational reasons to avoid misidentifying MVC during recovery periods. Total impulse was determined from the sum of impulses of all 60 MVC. Mean MVC force and EMG amplitudes were averaged across MVC within 10 consecutive bins of 6 MVC. End-test force reflecting the critical force threshold was defined as the mean MVC force of the last 6 MVC (bin 10). Fatigue indexes were calculated for mean MVC force and EMG amplitude, as the percentage decline from the first 6 (bin 1) to the last 6 MVC (bin 10). Explosive impulse over the first 0–150 ms from force onset (IMP0–150) was determined for each valid MVC using the same methods as that mentioned earlier for the unfatigued MVC, and averaged across the three valid MVC in each bin with the greatest IMP0–150. Explosive impulses over earlier phases (0–50 and 0–100 ms) were not calculated for the fatigue protocol because of the inherent variability in early-phase explosive force (13), which seemed augmented by our fatigue protocol likely due to the limited recovery time between MVC. Explosive RMS EMG amplitude over the first 0–150 ms from EMG onset (EMG0–150) was also measured and averaged across the same three MVC used to determine IMP0–150 in each bin. EMG onset was defined in the first muscle to be activated, as the last data point before the RMS EMG signal with a 2-ms moving time constant increased and remained above the mean of the baseline RMS for 0.5 s. Fatigue indexes from bin 1 to 10 were calculated as those mentioned previously for IMP0–150 and EMG0–150.
Tetanic peak forces and RFD at 10, 20, 50, and 100 Hz and the 20/50-Hz ratio were determined for the tetanic contractions after the fatigue protocol via the same methods explained earlier for the unfatigued conditions. The fatigue index (percentage decline) from pre– to post–fatigue protocol was determined for each frequency and the 20/50-Hz ratio for both tetanic peak force and RFD. A positive fatigue index for the 20/50 ratio (i.e., a decline in this ratio following the fatigue protocol) was considered evidence of low-frequency fatigue.
Plasma Nitrate and Nitrite
Capillary, finger-prick blood samples were taken upon arrival to the laboratory for each experimental trial. Whole blood was collected into 3 × 300 μL EDTA-treated microvettes and immediately centrifuged in a microcentrifuge for 15 min at 1000g. After centrifugation, the supernatant (300–400 μL) was removed and frozen at −80°C until analysis. Plasma nitrate and nitrite concentrations were determined by ozone-based chemiluminescence (model 88AM; Eco Physics) using previously reported methods (25). First, total NOx (all nitroso species) was measured by injecting an aliquot (50 μL) of each sample into a solution of vanadium (III) chloride (50 mM) dissolved in 1 M HCl, within an airtight microreaction vessel connected to the chemiluminescence analyser. Plasma nitrite was then determined in a two-step process by (i) injecting an aliquot (100 μL) of plasma into a solution of glacial acid acetic containing 45 mM potassium iodide and 10 mM iodide at 60°C, and actively purged by inert He, which measured plasma nitrite + other nitroso species (but not nitrate), and (ii) treating the plasma with acidic sulfanilamide (1 M HCl) to scavenge nitrite, before injection (100 μL), allowing for quantification of nitroso species not including nitrate or nitrite. Nitrite was then determined as the difference between the measures in step i and ii, whereas plasma nitrate was determined as the difference between total NOx and the measure in step i. Resources were only available to analyze plasma samples from the first 11 participants to complete the study.
Two-way repeated-measures ANOVA was used to determine the effects of supplementation on: unfatigued explosive impulse (two supplements (PLA and NIT) vs three time-epochs (IMP0–50, IMP0–100, and IMP0–150)); unfatigued normalized tetanic peak force and RFD (two supplements (PLA and NIT) vs three frequencies (10, 20, and 50 Hz)), and fatigue index of both tetanic peak force and RFD (two supplements (PLA and NIT) vs four frequencies (10, 20, 50, and 100 Hz)). In the instance of a main or interaction effect, paired t-tests were used for post hoc paired comparisons. The effects of supplementation on all other dependent variables were determined via paired t-tests. Cohen’s d effect sizes were determined for each paired comparison (26). To determine if mean MVC force had reached a plateau representing a critical torque threshold in the fatigue protocol, a linear function (y = mx + c) was fitted to the data plotting the relationship between mean MVC force (y) and MVC number (x) for the last 6 MVC (bin 10). The slope (m) of this linear function was compared with zero using a paired t-test, for PLA and NIT separately, with no significant differences reflecting a plateau in mean MVC force. Statistical significance was considered where P < 0.05, and statistical analysis was completed using IBM SPSS Statistics version 21. Data are reported as means ± SD.
Nitrate and Nitrite
Plasma nitrate and nitrite were 10.3-fold greater (P < 0.001; d = 12.6) and 1.8-fold greater (P = 0.002; d = 4.66), respectively, in NIT than in PLA (Fig. 1)
There was no effect of supplement on MVF (P = 0.887; d = 0.01; Table 1). There was also no main effect of supplementation (P = 0.911) or supplementation by time–epoch interaction effect (P = 0.903) on explosive impulse recorded during the MVC performed in unfatigued conditions (Table 1).
Tetanic peak force at 100 Hz in unfatigued conditions was 47% ± 4% of MVF and 46% ± 3% of MVF in PLA and NIT, respectively, with no difference in the absolute value between the two conditions (PLA: 348 ± 60 N; NIT: 343 ± 60 N; P = 0.176; d = 0.08). There was also no difference in tetanic peak RFD at 100 Hz between PLA (4468 ± 942 N·s−1) and NIT (4542 ± 982 N·s−1; P = 0.608; d = 0.08). These results suggest that stimulation intensity was constant across conditions. There were no main effects of supplementation (P ≥ 0.718) or supplementation by frequency interaction effects (P ≥ 0.382) on either tetanic peak force or RFD recorded at 10, 20, and 50 Hz, in unfatigued conditions (Table 1). Consequently, the 20/50-Hz ratio in unfatigued conditions was also similar between NIT and PLA for both tetanic peak force (P = 0.317; d = 0.16; Table 1) and RFD (P = 0.657; d = 0.10; Table 1).
Validity of fatigue protocol
Three participants achieved <90% MVF in the first MVC and/or <85% MVF in the second MVC of the fatigue protocol in either PLA and/or NIT, and so were excluded from all fatigued-condition measurements on the assumption that they were not performing maximal efforts from the start. In the remaining 14 participants, mean MVC force averaged across the 6 MVC within each consecutive bin, declined in an exponential manner (Fig. 2), so by the last 6 MVC (bin 10), the slope of the linear relationship between mean MVC force (relative to MVF) and MVC number was statistically similar to zero in both PLA (−0.17% ± 0.74% MVF/MVC; P = 0.394) and NIT (0.52% ± 1.23% MVF/MVC; P = 0.140). This suggests that the 14 remaining participants were consistently producing maximal efforts throughout the protocol, and mean MVC force declined to an asymptote likely representative of a critical force threshold (22). One participant of the 14 remaining was unable to record a valid MVC for explosive impulse analysis (i.e., there was countermovement or pretension before force onset) during the first 6 MVC (bin 1) in PLA, and so was removed from analysis of explosive impulse and EMG during the fatigue protocol.
There were no effects of supplementation on total impulse (P = 0.326; d = 0.05), end-test force (P = 0.388; d = 0.07), mean MVC force fatigue index (P = 0.198; d = 0.19), or mean MVC EMG amplitude fatigue index (P = 0.308; d = 0.21) recorded during the fatigue protocol (Table 2). However, the fatigue index for IMP0–150 was greater in the PLA compared with NIT for 8 of the 13 participants (Fig. 3) resulting in a moderate and statistically significant effect of supplementation on explosive impulse fatigue (P = 0.039; d = 0.51; Table 2; Fig. 3). This was despite a similar fatigue index for EMG0–150 in both conditions (P = 0.286; d = 0.39; Table 2).
There was a main effect of stimulation frequency on tetanic peak force fatigue index (P < 0.001) due to greater fatigue at 10 Hz than all other frequencies (P < 0.001; d = 0.62–1.27) and greater fatigue at 20 Hz than 50 or 100 Hz (P < 0.001; d = 0.60–0.76), whereas fatigue at 50 and 100 Hz was similar (P = 1. 000; d = 0.01–0.11), in both PLA and NIT (Fig. 4A). There was also a main effect of stimulation frequency on tetanic peak RFD (P < 0.001), with fatigue tending to be greater at 10 than 20 Hz (P = 0.011–0.094; d = 0.42–0.56), greater at both 10 and 20 Hz than 50 or 100 Hz (P < 0.001; d = 0.41–1.33), and greater at 50 than 100 Hz (P < 0.001; d = 0.43–0.54), in both PLA and NIT (Fig. 4B). The systematically greater fatigue at low (≤20 Hz) compared with high (≥50 Hz) frequencies resulted in reductions from pre– to post–fatigue protocol in the 20/50-Hz ratio for both peak force and peak RFD, in PLA and NIT (Figs. 4C, D), showing the occurrence of low-frequency fatigue in both conditions.
There was no main effect of supplementation (P = 0.615) or supplementation by frequency interaction effect (P = 0.253) on the fatigue index for tetanic peak force. Although there was also no main effect of supplementation on the fatigue index for peak RFD (P = 0.496), there was a supplementation by interaction effect (P = 0.042); however, paired comparisons showed no differences in peak RFD fatigue index between PLA and NIT at any of the frequencies (P ≥ 0.647; d = 0.05–0.27; Fig. 4B). Interestingly, there was a smaller reduction in the 20/50-Hz peak force ratio from pre– to post–fatigue protocol in NIT than in PLA for 12 of the 14 participants (Fig. 4C), resulting in a low-moderate effect of supplementation (d = 0.46) that did not reach statistical significance (P = 0.110). There was also a smaller reduction in the 20/50-Hz peak RFD ratio from pre– to post–fatigue protocol in NIT than in PLA (Fig. 4D) that was a large effect (d = 0.83) and did reach statistical significance (P = 0.011).
This was the first study to investigate the influence of dietary nitrate supplementation on voluntary and involuntary contractile performance in both unfatigued and fatigued conditions. We found no evidence of improved contractile performance in unfatigued conditions after nitrate supplementation, with PLA and NIT recording similar MVF, explosive voluntary impulse over all measured time periods, tetanic peak forces, and tetanic peak RFD at all stimulation frequencies. In contrast, nitrate supplementation reduced fatigue of voluntary explosive impulse by ~11%, during a bout of 60 MVC. Furthermore, low-frequency fatigue (reduction in 20/50-Hz ratio) was lower in NIT compared with PLA by ~28% and ~39% for tetanic peak force and RFD, respectively, despite fatigue indexes of peak force and RFD being statistically similar for NIT and PLA, at each separate frequency. This suggests that nitrate supplementation attenuated the disruption of excitation–contraction coupling caused by the fatiguing protocol, which might explain the reduced fatigue of voluntary explosive impulse in the NIT condition. The benefits of nitrate supplementation to voluntary force production in fatigued conditions were specific to the rising force–time curve, as total force–time impulse, end-test force, and mean MVC force fatigue index during the bout of 60 MVC were similar for NIT and PLA.
Plasma Nitrate and Nitrite
Seven days of nitrate supplementation successfully raised plasma nitrate and nitrite (10.3- and 1.8-fold, respectively) compared with the nitrate-depleted placebo. We believe that these changes measured in only 11 participants likely reflect the responses in all 17 participants, given consistent observations of raised plasma nitrate and nitrite after both acute (≤24 h) and chronic (2–15 d) nitrate supplementation with smaller doses than the present study (<12.9 mmol·d−1 [5,6,26–28]). The measured increase in plasma nitrite is of particular importance because this seems to be required to realize typical physiological benefits (e.g., reduced blood pressure) of nitrate supplementation (29). Dietary nitrate intake was not restricted in the current study, and so baseline (PLA) plasma nitrite (352 ± 61 nm) was comparable to other studies without dietary nitrate restrictions (~216–454 nm [6,21]), but higher than that recorded in studies with dietary nitrate restrictions (~80–331 nm [(5,27,28]). In addition to not restricting dietary nitrate, our blood sampling method (capillary blood in EDTA microvettes) differed from that of other studies (venous blood in lithium heparin tubes [5,6,21,27,28]), and this may also have contributed to baseline plasma nitrite values in the upper end of the range reported in the literature. Dietary nitrate intake was not restricted in the current study, consistent with the first human study to measure improved contractile performance with nitrate supplementation (9).
In unfatigued conditions, nitrate supplementation had no effect on tetanic peak forces or RFD at any stimulation frequency or on the 20/50-Hz ratios for peak force or RFD. These results are consistent with Hoon et al. (12), but in contrast to two recent studies reporting improvements in low-frequency (≤20 Hz) force (9,11), increased 20/50-Hz peak force ratio (9), and increased twitch and 300-Hz tetanic explosive forces (9) after nitrate supplementation. The current investigation and the three cited studies all tested the quadriceps muscles of seemingly similar cohorts of healthy, young, low/recreationally active men, and involved chronic (4–7 d) supplementation of relatively high doses of nitrate (>9.7 mmol·d−1), so the reasons for the inconsistent findings are unclear. Nevertheless, here we offer four possible explanations to help direct future research. (I) We and the three cited studies used beetroot juice for nitrate supplementation, but only the current study and Hoon et al. (12) compared the nitrate condition to a placebo of nitrate-depleted beetroot juice rather than blackcurrant cordial with lemon juice (9) or no placebo (11). Thus, other nutrients (e.g., polyphenols or antioxidants) in beetroot juice may have caused the effects observed by Haider and Folland (9) and Whitfield et al. (11). (II) On the basis of rodent models, it seems that nitrate supplementation only improves excitation–contraction coupling in fast-twitch fibers (10), so the contractile responses are likely to be diluted and variable in the mixed-fiber human quadriceps muscles, which are typically ~50% fast twitch (30) but can vary from 20% to 80% (31). (III) Increases in low-frequency force with nitrate supplementation seem to be negatively related to habitual dietary nitrate intake (9), so it is possible the habitual dietary nitrate intake of participants in the current study and that of Hoon et al. (12) was greater than that required to realize benefits from supplementation in unfatigued conditions. (IV) In a series of muscle contractions, such as those used in the methods of this and the other investigations, the first contraction(s) in the series would potentiate force and RFD of subsequent low-frequency contractions, through phosphorylation of myosin regulatory light chains increasing Ca2+ sensitivity of the myosin–actin cross-bridge (32). It is conceivable that potentiation may mask the benefits of nitrate supplementation in unfatigued conditions, and that the amount of potentiation differed between this and previous investigations, but because potentiation was not quantified in any of these studies, this remains speculative.
MVF was unaffected by supplementation, which is consistent with previous findings (9,12,21), and expected given that nitrate supplementation does not affect force at the firing frequencies (>20 Hz [9,11]) typically expected at MVF (~25–40 Hz [33,34]). Explosive impulse recorded over all measured time periods from force onset in explosive voluntary contractions in unfatigued conditions was also unaffected by nitrate supplementation in the current study. Theoretically, improved excitation–contraction coupling characterized by increased force at low frequencies—and thus low free cytosolic Ca2+—may benefit voluntary explosive force where cytosolic Ca2+ is rising. However, given that we found no evidence of improved excitation–contraction coupling in unfatigued conditions, it is no surprise that voluntary explosive impulse was also unaffected. Furthermore, nitrate supplementation does not seem to improve voluntary explosive force in unfatigued conditions, even when there is evidence of enhanced excitation–contraction coupling (9). The effects of nitrate supplementation on contractile performance might become more evident in fatigued conditions where excitation–contraction coupling is disrupted.
The fatigue protocol of 60 MVC resulted in a decline in mean MVC force to a plateau reflecting a critical threshold in both NIT and PLA. This threshold is thought to represent the highest force that can be maintained with metabolic steady state (22), which likely could not be overcome in the last 6 MVC of the protocol because of high-energy phosphate depletion and considerable metabolite accumulation (23). Thus, we are confident that a plateau in the fatigue and metabolic responses was reached in both conditions. As predicted, the 60 MVC provided sufficient force–time impulse and metabolic perturbation to elicit the first component of low-frequency fatigue (19,20), evidenced by greater fatigue at low (≤20 Hz) compared with high (≥50 Hz) frequencies, and a decline in 20/50-Hz ratios for tetanic peak force and RFD, in both NIT and PLA. This low-frequency fatigue reflects disruption of excitation–contraction coupling in both conditions, probably caused by a decline in SR Ca2+ release (16–18).
The disrupted excitation–contraction coupling likely contributed to the decline in voluntary explosive impulse from the first 6 to the last 6 MVC in both NIT and PLA, during the fatiguing protocol. Interestingly, nitrate supplementation attenuated the decline in voluntary explosive impulse, as evidenced by the significantly smaller fatigue index in NIT compared with PLA, although this effect was only observed in 8 of 13 participants, suggesting some variability in individual responses. Nevertheless, this study provides novel evidence that nitrate supplementation benefits explosive force production in fatigued but not unfatigued conditions. The reduced explosive impulse fatigue in NIT does not seem to be due to differences in neural drive between the conditions, as the explosive EMG fatigue index was similar for NIT and PLA. Therefore, mechanisms at the muscle, which may be associated with attenuated disruption of excitation–contraction coupling (discussed hereinafter), likely explain the effects of nitrate supplementation on explosive impulse fatigue index.
Nitrate supplementation did not affect total impulse, end-test force, or mean MVC force fatigue index recorded during the 60 MVC. Thus, the benefits of nitrate supplementation on voluntary force during fatiguing exercise seem to be specific to the rising slope of the force–time curve. This provides some evidence for attenuated disruption of the excitation–contraction coupling in NIT, which would theoretically have its greatest influence during contractile conditions of low or rising free cytosolic Ca2+, such as during the rising slope but not necessarily the plateau of MVC force–time curves. One other study (21) has assessed voluntary forces during repeated MVC (50 MVC) after nitrate supplementation and, similar to our results, found no differences in the MVC forces recorded over the plateau of the force–time curve, but they did not measure explosive forces during the rising slope.
Although the fatigue index for tetanic peak force at each frequency was similar for NIT and PLA, there was evidence of reduced low-frequency fatigue in NIT compared with PLA. Specifically, the decline in 20/50-Hz peak force ratio (a typical measure of low-frequency fatigue ) was ~28% lower in NIT compared with PLA. This difference was not statistically significant (P = 0.110), but there was a low-moderate effect (d = 0.46), and it occurred in 12 of 14 participants. The reduced low-frequency fatigue in NIT compared with PLA becomes more evident when considering the tetanic peak RFD results, where the decline in the 20/50-Hz ratio for peak RFD was ~39% lower after nitrate supplementation, which was a large and statistically significant effect (P = 0.011; d = 0.83). Furthermore, there was a supplementation by frequency interaction effect on tetanic peak RFD fatigue index, which seems to be due to the low frequencies (≤20 Hz) typically displaying less fatigue and the high frequencies (≥50 Hz) displaying more fatigue, in NIT compared with PLA (Fig. 4B), although paired differences at each frequency were not statistically significant. Collectively, these results provide novel evidence of reduced low-frequency fatigue after nitrate supplementation, which is more evident during the rising slope of the tetanic force–time curve compared with the peak, and suggests reduced disruption of the excitation–contraction coupling during fatiguing exercise. The first component of low-frequency fatigue seems dependent on metabolite accumulation causing reductions in SR Ca2+ release (16,17). In the current study, nitrate supplementation may have attenuated the first component of low-frequency fatigue by (i) lowering the PCr cost of force production (21) and thus blunting the metabolite accumulation thought to cause reduced Ca2+ release, and/or (ii) increasing SR Ca2+ release for a given excitation in the fast-twitch (fatiguing) fibers (10). The same mechanisms likely also explain the reduced fatigue index of voluntary explosive impulse during the 60 MVC after nitrate supplementation. Furthermore, although the current study assessed the first component of low-frequency fatigue, it is possible that nitrate supplementation—via mechanism ii—might similarly benefit the second component of low-frequency fatigue, which is also caused by reduced SR Ca2+ release, but independently of metabolite accumulation after several minutes of recovery (15,16,18).
In conclusion, we provide novel evidence that nitrate supplementation attenuates the reduction in explosive force production during fatiguing exercise, which seems likely due to attenuated disruption of excitation–contraction coupling, evidenced by decreased low-frequency fatigue in NIT compared with PLA. However, nitrate supplementation had no effect on voluntary or involuntary contractile performance in unfatigued conditions, which is in contrast to two recent studies showing increased low-frequency tetanic force after nitrate supplementation.
Explosive force production is functionally important where time to develop force is limited, such as during sprinting (36), joint stabilization (37), and balance recovery (38). Thus, the considerable declines in explosive force observed during fatiguing exercise (39) and match play (40) will greatly impair exercise performance and increase injury risk. In the current study, nitrate supplementation reduced the decline in explosive force during fatiguing exercise and so may benefit exercise performance and reduce the risk of injury during fatiguing activity where explosive contractions are required.
The authors would like to sincerely thank the following people for their help in producing this manuscript: Dr. Jonathan Folland (Loughborough University, for advice during the study design stage); Dr. Giulia Corona (University of Roehampton), for advice with plasma nitrate/nitrite analysis; Dr. Gunter Kuhnle (University of Reading) and Dr. Virag Sagi-Kiss (University of Reading), for completing the nitrate/nitrite analysis; and Rupert Manwaring, Holly Medlock, Elisa Giacomelli Ranzi, and Filipe Campos De Souza, for their help in the data collection.
This work was funded by the University of Roehampton.
The authors have no professional relationships with companies or manufacturers that may benefit from the results of the present study. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The authors declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.
1. Moncada S, Higgs A. The L-arginine–nitric oxide pathway. N Engl J Med
2. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov
3. Lundberg JO, Carlstrom M, Larsen FJ, Weitzberg E. Roles of dietary inorganic nitrate in cardiovascular health and disease. Cardiovasc Res
4. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med
5. Bailey SJ, Winyard P, Vanhatalo A, et al. Dietary nitrate supplementation reduces the O2
cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol (1985)
6. Vanhatalo A, Bailey SJ, Blackwell JR, et al. Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am J Physiol Regul Integr Comp Physiol
7. Cermak NM, Gibala MJ, van Loon LJ. Nitrate supplementation’s improvement of 10-km time-trial performance in trained cyclists. Int J Sport Nutr Exerc Metab
8. Jones AM. Influence of dietary nitrate on the physiological determinants of exercise performance: a critical review. Appl Physiol Nutr Metab
9. Haider G, Folland JP. Nitrate supplementation enhances the contractile properties of human skeletal muscle. Med Sci Sports Exerc
10. Hernandez A, Schiffer TA, Ivarsson N, et al. Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle. J Physiol
11. Whitfield J, Gamu D, Heigenhauser GJF, et al. Beetroot juice
increases human muscle force without changing Ca2+
-handling proteins. Med Sci Sports Exerc
12. Hoon MW, Fornusek C, Chapman PG, Johnson NA. The effect of nitrate supplementation on muscle contraction in healthy adults. Eur J Sport Sci
13. Folland JP, Buckthorpe MW, Hannah R. Human capacity for explosive force production: neural and contractile determinants. Scand J Med Sci Sports
14. Tillin NA, Jimenez-Reyes P, Pain MT, Folland JP. Neuromuscular performance of explosive power athletes versus untrained individuals. Med Sci Sports Exerc
15. Edwards RH, Hill DK, Jones DA, Merton PA. Fatigue of long duration in human skeletal muscle after exercise. J Physiol
16. Chin ER, Balnave CD, Allen DG. Role of intracellular calcium and metabolites in low-frequency fatigue
of mouse skeletal muscle. Am J Physiol
. 1997;272(2 Pt 1):C550–9.
17. Hill CA, Thompson MW, Ruell PA, Thom JM, White MJ. Sarcoplasmic reticulum function and muscle contractile character following fatiguing exercise in humans. J Physiol
. 2001;531(Pt 3):871–8.
18. Westerblad H, Duty S, Allen DG. Intracellular calcium concentration during low-frequency fatigue
in isolated single fibers of mouse skeletal muscle. J Appl Physiol (1985)
19. Binder-Macleod SA, Russ DW. Effects of activation frequency and force on low-frequency fatigue
in human skeletal muscle. J Appl Physiol (1985)
20. Griffin L, Anderson NC. Fatigue in high- versus low-force voluntary and evoked contractions. Exp Brain Res
21. Fulford J, Winyard PG, Vanhatalo A, Bailey SJ, Blackwell JR, Jones AM. Influence of dietary nitrate supplementation on human skeletal muscle metabolism and force production during maximum voluntary contractions. Pflugers Arch
22. Burnley M. Estimation of critical torque using intermittent isometric maximal voluntary contractions of the quadriceps in humans. J Appl Physiol (1985)
23. Burnley M, Vanhatalo A, Fulford J, Jones AM. Similar metabolic perturbations during all-out and constant force exhaustive exercise in humans: a (31)P magnetic resonance spectroscopy study. Exp Physiol
24. Maffiuletti NA, Aagaard P, Blazevich AJ, Folland J, Tillin N, Duchateau J. Rate of force development
: physiological and methodological considerations. Eur J Appl Physiol
25. Rendeiro C, Dong H, Saunders C, et al. Flavanone-rich citrus beverages counteract the transient decline in postprandial endothelial function in humans: a randomised, controlled, double-masked, cross-over intervention study—corrigendum. Br J Nutr
26. Cohen J. Statistical Power Analysis for the Behavioural Sciences
. ed. Hillsdale (NJ): Lawrence Erlbaum Associates; 1988. pp. 21.
27. Wylie LJ, Kelly J, Bailey SJ, et al. Beetroot juice
and exercise: pharmacodynamic and dose–response relationships. J Appl Physiol (1985)
28. Christensen PM, Petersen NK, Friis SN, Weitzberg E, Nybo L. Effects of nitrate supplementation in trained and untrained muscle are modest with initial high plasma nitrite levels. Scand J Med Sci Sports
29. Webb AJ, Patel N, Loukogeorgakis S, et al. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension
30. Johnson MA, Polgar J, Weightman D, Appleton D. Data on the distribution of fibre types in thirty-six human muscles. an autopsy study. J Neurol Sci
31. Andersen JL. Muscle fibre type characteristics of the runner In: Bangsbo J, Larsen HB, editors. Running & Science in an Inter-Disciplinary Perspective
. Copenhagen: Munksgaard Publishing; 2001:49–65.
32. Tillin NA, Bishop D. Factors modulating post-activation potentiation and its effect on performance of subsequent explosive activities. Sports Med
33. Pucci AR, Griffin L, Cafarelli E. Maximal motor unit firing rates during isometric resistance training in men. Exp Physiol
34. Knight CA, Kamen G. Relationships between voluntary activation and motor unit firing rate during maximal voluntary contractions in young and older adults. Eur J Appl Physiol
35. Keeton RB, Binder-Macleod SA. Low-frequency fatigue
. Phys Ther
36. Tillin NA, Pain MT, Folland J. Explosive force production during isometric squats correlates with athletic performance in rugby union players. J Sports Sci
37. Krosshaug T, Nakamae A, Boden BP, et al. Mechanisms of anterior cruciate ligament injury in basketball: video analysis of 39 cases. Am J Sports Med
38. Izquierdo M, Aguado X, Gonzalez R, Lopez JL, Hakkinen K. Maximal and explosive force production capacity and balance performance in men of different ages. Eur J Appl Physiol Occup Physiol
39. Buckthorpe M, Pain MT, Folland JP. Central fatigue contributes to the greater reductions in explosive than maximal strength with high-intensity fatigue. Exp Physiol
40. Thorlund JB, Aagaard P, Madsen K. Rapid muscle force capacity changes after soccer match play. Int J Sports Med