Muscle metaboreflex activation during submaximal dynamic exercise, increases arterial pressure primarily via increases in cardiac output as there is little systemic vasoconstriction. Indeed, in normal animals we have often shown a small, but significant, peripheral vasodilation during metaboreflex activation, which is mediated, at least in part, by release of epinephrine and activation of vascular β₂ receptors. We tested whether this vasodilation is in part due to increased release of nitric oxide caused by the rise in cardiac output eliciting endothelium-dependent flow-mediated vasodilation. The muscle metaboreflex was activated via graded reductions in hindlimb blood flow during mild exercise with and without nitric oxide synthesis blockade (L-nitroarginine methyl ester (L-name); 5mg/kg). We assessed the role of increased cardiac output in mediating peripheral vasodilation via the slope of the relationship between the rise in non-ischemic vascular conductance (conductance of all vascular beds excluding hindlimbs) vs. the rise in cardiac output. L-name increased mean arterial pressure at rest and during exercise. The metaboreflex-induced increases in mean arterial pressure were unaltered by L-name; whereas, the increases in cardiac output and non-ischemic vascular conductance were attenuated. However, the slope of the relationship between non-ischemic vascular conductance and cardiac output was not affected by L-name indicating that the rise in cardiac output did not elicit vasodilation via increased release of nitric oxide. Thus, although nitric oxide is intrinsic to the vascular tonus, endothelial-dependent flow-mediated vasodilation plays little role in the small peripheral vasodilation observed during muscle metaboreflex activation.