Although spaceflight and bed rest are known to cause muscular atrophy in the anti-gravity muscles of the legs, the changes in sympathetic and cardiovascular responses to exercises using the atrophied muscles remain unknown. We hypothesized that bed rest would augment sympathetic responses to isometric exercise using anti-gravity leg muscles in humans. Ten healthy male volunteers were subjected to 14-day 6° head-down bed rest. Before and after bed rest, they performed isometric exercises using leg (plantar flexion) and forearm (handgrip) muscles, followed by 2-min post-exercise muscle ischemia (PEMI) that continues to stimulate the muscle metaboreflex. These exercises were sustained to fatigue. We measured muscle sympathetic nerve activity (MSNA) in the contralateral resting leg by microneurography. In both pre- and post-bed rest exercise tests, exercise intensities were set at 30% and 70% of the maximum voluntary force measured before bed rest. Bed rest attenuated the increase in MSNA in response to fatiguing plantar flexion by approximately 70% at both exercise intensities (both, P<0.05 vs. before bed rest), and reduced the maximal voluntary force of plantar flexion by 15%. In contrast, bed rest did not alter the increase in MSNA response to fatiguing handgrip, and had no effects on the maximal voluntary force of handgrip. Although PEMI sustained MSNA activation before bed rest in all trials, bed rest entirely eliminated the PEMI-induced increase in MSNA in leg exercises but partially attenuated it in forearm exercises. These results do not support our hypothesis, but indicate that bed rest causes a reduction in isometric exercise-induced sympathetic activation in (probably atrophied) anti-gravity leg muscles.