Previous studies have suggested that melatonin alters sympathetic outflow in humans. The purpose of the present study was to determine in humans the effect of melatonin on sympathetic nerve activity and arterial blood pressure during orthostatic stress. Fifty minutes after receiving a 3 mg tablet of melatonin or placebo (different days), muscle sympathetic nerve activity (MSNA), arterial blood pressure, heart rate, forearm blood flow and thoracic impedance were measured for 10 min at rest and during 5 min of lower body negative pressure (LBNP) at -10 and -40 mmHg (n= 11). During LBNP, MSNA responses were attenuated after melatonin at both -10 and -40 mmHg (P < 0.03). Specifically, during the placebo trial, MSNA increased by 33 ± 8 and 251 ± 70 % during -10 and -40 mmHg, respectively, but increased by only 8 ± 7 and 111 ± 35 % during -10 and -40 mmHg with melatonin, respectively. However, arterial blood pressure and forearm vascular resistance responses were unchanged by melatonin during LBNP. MSNA responses were not affected by melatonin during an isometric handgrip test (30 % maximum voluntary contraction) and a cold pressor test. Plasma melatonin concentration was measured at 25 min intervals for 125 min in six subjects. Melatonin concentration was 14 ± 11 pg ml−1 before ingestion and was significantly increased at each time point (peaking at 75 min; 1830 ± 848 pg ml−1). These findings indicate that in humans, a high concentration of melatonin can attenuate the reflex sympathetic increases that occur in response to orthostatic stress. These alterations appear to be mediated by melatonin-induced changes to the baroreflexes.