Spaceflight causes sensorimotor adaptations that result in balance deficiencies on return to a gravitational environment. Treadmill exercise within lower-body negative pressure (LBNP) helps protect physiological function during microgravity as simulated by bed rest. Therefore, we hypothesized that treadmill exercise within LBNP would prevent balance losses in both male and female identical twins during 30 days of 6° head-down tilt bed rest. Fifteen (seven female and eight male) identical twin sets participated in this simulation of microgravity. Within each twin pair, one twin was randomly assigned to an exercise group that performed 40 min of supine treadmill exercise within LBNP set to generate 1.0-1.2 body weight, followed by 5 min of static feet-supported LBNP, 6 days per week. Their identical sibling was assigned to a non-exercise control group with all other bed rest conditions equivalent. Before and immediately after bed rest, subjects completed standing and walking rail balance tests with eyes open and eyes closed. In control subjects, standing rail balance times (men: -42%, women: -40%), rail walk distances (men: -44%, women: -32%) and rail walk times (men: -34%, women: -31%) significantly decreased after bed rest. Compared with controls, treadmill exercise within LBNP significantly attenuated losses of standing rail balance time by 63% in men, but the 41% attenuation in women was not significant. Treadmill exercise within LBNP did not affect rail walk abilities in men or women. Treadmill exercise within LBNP during simulated spaceflight attenuates loss of balance control in men but not in women.