To test the hypothesis that the magnitude of reduction in plasma volume and work capacity following exposure to simulated microgravity is dependent on the initial level of aerobic fitness, peak oxygen uptake (V˙o2peak) was measured in a group of physically fit subjects and compared with V˙o2peak in a group of relatively unfit subjects before and after 10 days of continuous 6° head-down tilt (HDT). Ten fit subjects (40 ± 2 year) with mean ± SE V˙o2peak = 48.9 ± 1.7 mL kg−1 min−1 were matched for age, height, and lean body weight with 10 unfit subjects (V˙o2peak = 37.7 ± 1.6 mL kg−1 min−1). Before and after HDT, plasma, blood, and red cell volumes and body composition were measured and all subjects underwent a graded supine cycle ergometer test to determine V˙o2peak period needed. Reduced V˙o2peak in fit subjects (−16.2%) was greater than that of unfit subjects (−6.1%). Similarly, reductions in plasma (−18.3%) and blood volumes (−16.0%) in fit subjects were larger than those of unfit subjects (blood volume = −5.6%; plasma volume = −6.6%). Reduced plasma volume was associated with greater negative body fluid balance during the initial 24 h of HDT in the fit group (912 ± 154 mL) compared with unfit subjects (453 ± 200 mL). The percentage change for V˙o2peak correlated with percentage change in plasma volume (r = +0.79). Following exposure to simulated microgravity, fit subjects demonstrated larger reductions in V˙o2peak than unfit subjects which was associated with larger reductions in plasma and blood volume. These data suggest that the magnitude of physical deconditioning induced by exposure to microgravity without intervention of countermeasures was influenced by the initial fitness of the subjects.