Introduction: Chronic exposure to weightlessness induces a persistent headward fluid shift, which may contribute to the development of Spaceflight Associated Neuro-ocular Syndrome (SANS). Strict 6° head-down tilt bedrest (HDTBR) is a spaceflight analog that induces a headward fluid shift and development of SANS findings, enabling the evaluation of potential countermeasures. Lower body exercise can induce a sustained post-exercise hyperemia thereby shifting blood away from the central circulation. In addition, veno-constrictive thigh cuffs (VTC) can sequester blood volume in the legs. The purpose of this study was to determine whether 6 hours of daily VTC use following cycling exercise (Ex+VTC) could reverse the headward fluid shift during 30 days of strict 6° HDTBR. We hypothesized that Ex+VTC would sequester fluid in the legs leading to increased thigh circumference and reduced stroke volume (SV), cardiac output (CO), internal jugular vein (IJV) cross-sectional area (CSA) and pressure, and intraocular pressure (IOP). Methods: Participants were randomly assigned to a Control (n=12, 6 female) or Ex+VTC (n=12, 4 female) group and completed baseline data collection (BDC) in the seated and supine posture followed by 30 days of strict 6° HDTBR. The Ex+VTC group completed a maximal exercise test on a cycle ergometer in 6° HDT posture during BDC to establish work rates during HDTBR. The Ex+VTC group performed 10 minutes of light cycle exercise, cycled for 45 minutes at 45% of their max wattage, and then donned VTC to ~50 mmHg for 6 hours on 6 of 7 days per week during HDTBR. Data were collected before exercise and 30 minutes and 6 hours after exercise and donning VTCs on days 3, 17, and 29 of HDTBR. Controls were studied at similar time points. Days were nested within the models. Mixed models were used to determine differences between groups in the change in thigh circumference measured using a flexible tape measure, bilateral IOP measured using tonometry, CO measured using the inert gas rebreathe technique (Innocor), and ultrasound of bilateral IJV CSA and pressure from before to 30 min and 6 hours after use of the countermeasure. Left and right IJV and IOP data were nested within each participant. Results: The Ex+VTC group completed all exercise sessions followed by 6 hours of VTC use. Compared to Control, thigh circumference was greater after 30 minutes (+1.3 vs. -0.2 cm, p<0.001) and 6 hours of VTC use (+2.5 cm vs. -0.1 cm in controls, p<0.001). SV (-38 vs. 2 ml, p<0.001) and CO (-1.5 vs. 0.3 L/min, p<0.001) were reduced at 30 minutes and 6 hours of VTC use (SV: -30 vs. 5 ml, p<0.001; CO: -1.1 vs. 0.6 L/min, p<0.001) compared to Control. IJV CSA and pressure were reduced at 30 minutes (CSA, p<0.001; pressure, p=0.001) and 6 hours of VTC use (CSA, p<0.001; pressure, p=0.007). IOP was not different compared to Control. Conclusions:Use of VTC for 6 hours after 45 minutes of exercise successfully sequestered fluid in the legs, leading to a reduction in central blood volume, IJV CSA, and IJV pressure. Thus, an Ex+VTC countermeasure resulted in a partial fluid shift reversal. This research was supported by the NASA Human Research Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.