Humans exposed to real or simulated microgravity experience decrements in blood pressure regulation during orthostatic stress that may be related to autonomic dysregulation and/or hypovolemia. We examined the hypothesis that hypovolemia, without the deconditioning effects of bed rest or spaceflight, would augment the sympathoneural and vasomotor response to graded orthostatic stress. Radial artery blood pressure (tonometry), stroke volume (SV), brachial blood flow (Doppler ultrasound), heart rate (electrocardiogram), peroneal muscle sympathetic nerve activity (MSNA; microneurography), and estimated central venous pressure (CVP) were recorded during five levels (-5, -10, -15, -20 and -40 mmHg) of randomly assigned lower body negative pressure (LBNP) (n = 8). Forearm (FVR) and total peripheral vascular resistance (TPR) were calculated. The test was repeated under randomly assigned placebo (normovolemia) or diuretic (spironolactone: 100 mg/day, 3 days) (hypovolemia) conditions. The diuretic produced an approximately 16% reduction in plasma volume. Compared with normovolemia, SV and cardiac output were reduced by approximately 12% and approximately 10% at baseline and during LBNP after the diuretic. During hypovolemia, there was an upward shift in the %DeltaMSNA/DeltaCVP, DeltaFVR/DeltaCVP, and DeltaTPR/DeltaCVP relationships during 0 to -20 mmHg LBNP. In contrast to normovolemia, blood pressure increased at -40 mmHg LBNP during hypovolemia due to larger gains in the %DeltaMSNA/DeltaCVP and DeltaTPR/DeltaCVP relationships. It was concluded that acute hypovolemia augmented the neurovascular component of blood pressure control during moderate orthostasis, effectively compensating for decrements in SV and cardiac output.