Cardiovascular deconditioning after long duration spaceflight is especially challenging in women who have a lower orthostatic tolerance (OT) compared with men. We hypothesized that an exercise prescription, combining supine aerobic treadmill exercise in a lower body negative pressure (LBNP) chamber followed by 10 min of resting LBNP, three to four times a week, and flywheel resistive training every third day would maintain orthostatic tolerance (OT) in women during a 60-day head-down-tilt bed rest (HDBR). Sixteen women were assigned to two groups (exercise, control). Pre and post HDBR OT was assessed with a tilt/LBNP test until presyncope. OT time (mean +/- SE) decreased from 17.5 +/- 1.0 min to 9.1 +/- 1.5 min (-50 +/- 6%) in control group (P < 0.001) and from 19.3 +/- 1.3 min to 13.0 +/- 1.9 min (-35 +/- 7%) in exercise group (P < 0.001), with no significant difference in OT time between the two groups after HDBR (P = 0.13). Nevertheless, compared with controls post HDBR, exercisers had a lower heart rate during supine rest (mean +/- SE, 71 +/- 3 vs. 85 +/- 4, P < 0.01), a slower increase in heart rate and a slower decrease in stroke volume over the course of tilt/LBNP test (P < 0.05). Blood volume (mean +/- SE) decreased in controls (-9 +/- 2%, P < 0.01) but was maintained in exercisers (-4 +/- 3%, P = 0.17).Our results suggest that the combined exercise countermeasure did not significantly improve OT but protected blood volume and cardiovascular response to sub tolerance levels of orthostatic stress.