Lower body negative pressure (LBNP) simulates orthostatic stress by redistributing blood to the lower limbs, triggering cardiovascular compensatory responses without the influence of standing hydrostatic pressures. While central adaptations to LBNP are well-studied, lower limb vascular responses remain less characterized due to methodological constraints. This study examined lower limb vascular responses to LBNP and whether prior acute supine cycling influences blood flow regulation. We hypothesized that LBNP would increase deep femoral vein (DFV) but not the superficial femoral artery (SFA) diameter, decrease leg blood flow, increase calf volume, and that prior cycling would attenuate fluid shift-induced vascular changes via enhanced systemic arterial vasoconstriction and local arterial vasodilation. Twenty healthy adults (10 females, 25±4yrs) completed a randomized crossover protocol with a 15-minute rest condition and a 15-minute postexercise condition (cycling at heart rate of ~90 to 110bpm), separated by 15 minutes of rest. LBNP was applied at -20 mmHg and -40 mmHg, with vascular measures taken in the final minute of each stage. DFV (p=0.003) and SFA p0.05) except for a main effect on SFA blood flow (p=0.004), consistent with post-exercise hyperemia. These findings suggest LBNP reduces lower limb blood flow, induces venous distension, and increases calf volume, with minimal impact from acute exercise. Future research should explore the impact of higher-intensity exercise on LBNP-induced vascular responses.