Purpose: The purpose of this study is to characterize both the efficacy of altered restriction pressures and consequences of optimized blood flow restriction (BFR) for altering the cardiovascular and fluid regulating response in humans.Methods: This work comprised a series of related trials. Phase 1: during semi-recumbent cycling (5 min, 60 W) with BFR at 0%, 50%, 75%, 100%, and 125% of participants' lowest arterial occlusion pressure (LOP) echocardiographic images were collected alongside continuous heart rate (HR) and blood pressure (BP). In phase 2, 24 h fluid balance (intake-output) and fluid-regulating hormone responses were measured after a practical BFR exercise session (treadmill walking: 5<middle dot>3 min, 1 min rest, 5 km<middle dot>h -1 , 5% incline, 100% LOP). Phase 3 examined the magnitude and effect of blood volume distribution following BFR treadmill walking using a modified CO-rebreathe technique.Results: Cardiac ejection fraction remained stable irrespective of cuff pressure and despite a reduction in end-diastolic volume (Delta of 11 +/- 9 mL, P = 0.02). HR and BP were highest at 100% LOP (Delta HR = 18 +/- 19 bpm, Delta SBP = 51 +/- 30 mm Hg, Delta DBP = 33 +/- 15 mm Hg, Delta Rate Pressure Product = 76 +/- 32 bpm<middle dot>mm Hg<middle dot>100 -1 ). BFR treadmill walking stimulated a greater release of fluid-regulating hormones than normal walking (Delta renin = BFR: 25.3 +/- 24.2 vs CON: 9.1 +/- 11.8 ng<middle dot>L -1 ; Delta copeptin = BFR: 50.1 +/- 97.9 vs. CON: 0.43 +/- 0.8 pmol<middle dot>L -1 ), but no difference in fluid retention was observed ( P = 0.2). Approximately 27% of total blood volume was confined to the legs during BFR, but the relative volume trapped in the legs was not related to fluid-regulating hormone release (renin r = -0.04, P = 0.9; copeptin r = 0.27, P = 0.3).Conclusions: BFR exercise elicits the largest cardiovascular effect using 100% LOP, with evidence of appreciable fluid regulating hormonal response during a typical BFR exercise session. The relative volume of blood sequestered in the limb does not appear to drive this response.