The extent to which dietary supplementation with the nitric oxide synthase (NOS) substrate, L-arginine (ARG), impacts on NO production and NO-mediated physiological responses is controversial. This randomised, double blinded, cross-over study investigated the effects of acute ARG supplementation on NO biomarkers, O₂ cost of exercise and exercise tolerance in humans. In one experiment, 15 subjects completed moderate- and severe-intensity running bouts after acute supplementation with 6 g ARG or placebo (PLA). In another experiment, eight subjects completed moderate- and severe-intensity cycling bouts after acute supplementation with 6 g ARG plus 25 g of carbohydrate (ARG + CHO) or an energy-matched dose of carbohydrate alone (CHO). The plasma nitrite concentration was not different after ARG (Pre: 204 ± 79; Post: 241 ± 114 nM; P > 0.05) or ARG + CHO consumption (Pre: 304 ± 57; Post: 335 ± 116 nM; P > 0.05). During moderate-intensity exercise, the steady-state pulmonary VO₂ was not different, relative to the respective placebo conditions, after ARG (PLA: 2,407 ± 318, ARG: 2,422 ± 333 mL min(-1)) or ARG + CHO (CHO: 1,695 ± 304, ARG + CHO: 1,712 ± 312 mL min(-1)) ingestion (P > 0.05). The tolerable duration of severe exercise was also not significantly different (P > 0.05) after ingesting ARG (PLA: 551 ± 140, ARG: 552 ± 150 s) or ARG + CHO (CHO: 457 ± 182, ARG + CHO: 441 ± 221 s). In conclusion, acute dietary supplementation with ARG or ARG + CHO did not alter biomarkers of NO synthesis, O₂ cost of exercise or exercise tolerance in healthy subjects.