Acute dietary nitrate (NO3-) supplementation with beetroot juice can decrease blood pressure (BP) at rest (1) and during cycling exercise (2) in healthy normotensives. The mechanism responsible for these hypotensive responses is attributed to increased systemic levels of the potent vasodilator, nitric oxide (1-3). However, evidence also supports a central sympathoinhibitory role of nitric oxide (4). We hypothesized that acute beetroot juice supplementation would decrease BP at rest and during exercise, coincident with reductions in muscle sympathetic nerve activity (MSNA). Fourteen healthy normotensive participants (7 female; 25±10 years; 104±6/64±6 mmHg) completed a prospective, randomized, double blind, placebo-controlled crossover study comparing the acute effects of high-NO3- (70mL; ~6-8 mmol NO3-) and NO3–depleted placebo (70mL; ~4.8 μmole NO3-) beetroot juice supplementation (James White Drinks Ltd, UK) on BP and MSNA. Resting supine measurements of heart rate (ECG), minute-to-minute brachial BP (BpTRU), and calf MSNA (microneurography) were collected in 10 minute epochs at baseline and 3 hours post-ingestion (to coincide with peak plasma NO3-). Following the resting assessment, 11 participants underwent continuous supine measurements of BP (Finometer) and MSNA during a 3 minute baseline and 2 minute static handgrip exercise at 30% maximal voluntary contraction. All variables were assessed as the change from baseline (Δ) and compared using paired t-tests (placebo vs. NO3-); values presented as mean ± SD. Compared to placebo, resting systolic BP (Δ2±5 vs. -1±5 mmHg, p<0.05), and MSNA burst frequency (Δ3±4 vs. -3±5 bursts/min, p<0.01) and burst incidence (Δ4±5 vs. -4±7 bursts/100 heartbeats, p<0.01) were lower following NO3- supplementation, whereas diastolic BP (Δ5±7 vs. 4±5 mmHg, p>0.05) and heart rate (Δ-1±4 vs. 0±4 bpm, p>0.05) were unchanged. During static handgrip, systolic (Δ12±8 vs. 11±7 mmHg, p>0.05) and diastolic (Δ11±4 vs. 11±4 mmHg, p>0.05) BP and heart rate (Δ12±12 vs. 13±10 bpm; p>0.05) responses were similar between placebo and NO3- supplementation. In contrast, compared to placebo, MSNA burst frequency (Δ11±10 vs. 6±6 bursts/min, p<0.05) and burst incidence (Δ8±9 vs. 1±8 bursts/100 heartbeats, p<0.05) responses were attenuated following NO3- supplementation. Collectively, these data demonstrate in healthy normotensive participants that acute NO3- supplementation with beetroot juice can decrease central sympathetic outflow to skeletal muscle at rest and during exercise. This provides the first evidence for a potential neural contribution to the reported improvements in resting hemodynamics, endothelial function, and exercise tolerance. Dietary NO3- supplementation may provide a novel treatment for targeting exaggerated sympathoexcitation in cardiovascular disease states (e.g. heart failure).