Supine weight-bearing exercise within lower body negative pressure (LBNP) alleviates some of the skeletal deconditioning induced by simulated weightlessness in men. We examined this potential beneficial effect in women. Because dietary acid load affected the degree of bone resorption in men during bed rest, we also investigated this variable in women. Subjects were 7 pairs of female identical twins assigned at random to 2 groups, sedentary bed rest (control) or bed rest with supine treadmill exercise within LBNP. Dietary intake was controlled and monitored. Urinary calcium and markers of bone resorption were measured before bed rest and on bed rest days 5/6, 12/13, 19/20, and 26/27. Bone mineral content was assessed by dual-energy X-ray absorptiometry before and after bed rest. Data were analyzed by repeated-measures two-way analysis of variance. Pearson correlation coefficients were used to define the relationships between diet and markers of bone metabolism and to estimate heritability of markers. During bed rest, all markers of bone resorption and urinary calcium and phosphorus increased (P<0.001); parathyroid hormone (P=0.06), bone-specific alkaline phosphatase (P=0.06), and 1,25-dihydroxyvitamin D (P=0.09) tended to decrease. LBNP exercise tended to mitigate bone density loss. The ratio of dietary animal protein to potassium was positively correlated with urinary calcium excretion for all weeks of bed rest in the control group, but only during weeks 1 and 3 in the exercise group. Pre-bed rest data suggested that many markers of bone metabolism have strong genetic determinants. Treadmill exercise within LBNP had less of a protective effect on bone resorption during bed rest in women than previously published results had shown for its effect in men, but the same trends were observed for both sexes. Dietary acid load of these female subjects was significantly correlated with calcium excretion but not with other bone resorption markers.