Astronauts returning to Earth have reduced orthostatic: tolerance and exercise capacity. Alterations in autonomic nervous system and neuromuscular function after spaceflight might contribute to this problem. In this study, we tested the hypothesis that exposure to microgravity impairs autonomic neural control of sympathetic outflow in response to peripheral afferent stimulation produced by handgrip and a cold pressor test in humans. We studied five astronauts ∼72 and 23 days before, and on landing day after the 16 day Neurolab (STS-90) space shuttle mission, and four of the astronauts during flight (day 12 or 13). Heart rate, arterial pressure and peroneal muscle sympathetic nerve activity (MSNA) were recorded before and during static handgrip sustained to fatigue at 40% of maximum voluntary contraction, followed by 2 min of circulatory arrest pre-, in-and post-flight. The cold pressor test was applied only before (five astronauts) and during flight (day 12 or 13, four astronauts). Mean (± S.E.M.) baseline heart rates and arterial pressures were similar among pre-, in- and post-flight measurements. At the same relative fatiguing force, the peak systolic pressure and mean arterial pressure during static handgrip were not different before, during and after spaceflight. The peak diastolic pressure tended to be higher post-than pre-flight (112 ± 6 vs. 99 ± 5 mmHg, P = 0.088). Contraction-induced rises in heart rate were similar pre-, in- and post-flight. MSNA was higher post-flight in all subjects before static handgrip (26 ± 4 post- vs. 15 ± 4 bursts min-1 pre-flight, P = 0.017). Contraction-evoked peak MSNA responses were not different before, during, and after spaceflight (41 ± 4, 38 ± 5 and 46 ± 6 bursts min-1, all P > 0.05). MSNA during post-handgrip circulatory arrest was higher post- than pre- or in-flight (41 ± 1 vs. 33 ± 3 and 30 ± 5 bursts min-1, P = 0.038 and 0.036). Similarly, responses of MSNA and blood pressure to the cold pressor test were well maintained in-flight. We conclude that modulation of muscle sympathetic neural outflow by muscle metaboreceptors and skin nociceptors is preserved during short duration spaceflight.