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The purpose of this study is to assess cardiovascular responses to lower body positive pressure (LBPP) and to examine the effects of LBPP unloading on gait mechanics during treadmill ambulation. We hypothesized that LBPP allows comfortable unloading of the body with minimal impact on the cardiovascular system and gait parameters. Fifteen healthy male and female subjects (22–55 yr) volunteered for the study. Nine underwent noninvasive cardiovascular studies while standing and ambulating upright in LBPP, and six completed a gait analysis protocol. During stance, heart rate decreased significantly from 83 ± 3 beats/min in ambient pressure to 73 ± 3 beats/min at 50 mmHg LBPP ( P < 0.05). During ambulation in LBPP at 3 mph (1.34 m/s), heart rate decreased significantly from 99 ± 4 beats/min in ambient pressure to 84 ± 2 beats/min at 50 mmHg LBPP ( P < 0.009). Blood pressure, brain oxygenation, blood flow velocity through the middle cerebral artery, and head skin microvascular blood flow did not change significantly with LBPP. As allowed by LBPP, ambulating at 60 and 20% body weight decreased ground reaction force ( P < 0.05), whereas knee and ankle sagittal ranges of motion remained unaffected. In conclusion, ambulating in LBPP has no adverse impact on the systemic and head cardiovascular parameters while producing significant unweighting and minimal alterations in gait kinematics. Therefore, ambulating within LBPP is potentially a new and safe rehabilitation tool for patients to reduce loads on lower body musculoskeletal structures while preserving gait mechanics.