Astronauts develop Space flight-associated neuro-ocular syndrome due to the persistent lack of Earth’s gravity. At present, the pathophysiology is uncertain, but may be due to the absence of diurnal, postural reductions in intracranial pressure relative to intraocular pressure during periods of zero gravity (Lawley et al. 2017; Anderson et al. 2015). Thus, recreating gravitational gradients in space may be an effective countermeasure. We have recently shown that short durations (5 to 20 minutes) of low level (-20-30 mmHg) lower body negative pressure (LBNP) consistently reduces intracranial pressure by ~4 mmHg, and thus reintroduces about 25% of the gravitational effect of standing upright on earth (Petersen et al. 2018). The objective of this study was to determine if prolonged (8 hrs) simulated partial hydrostatic unloading of the brain via low-level LBNP could act as a countermeasure for choroidal engorgement associated with simulated microgravity by -6 deg head down tilt bedrest. This interventional study included five men and two women who spent 3 days in -6 deg head down tilt bedrest on two occasions in a randomized, counter-balanced, cross-over design. During one visit, participants spent 8 hours per day (10am – 6pm) inside an airtight chamber sealed at the level of the iliac crest with an internal pressure of -20 mmHg. Optical coherence tomography was performed in both trials in the supine position and after 3 days in -6 deg head down tilt. Moreover, in two participants intracranial pressure was measured directly for 9 hours in both trials to document the long-term effects of LBNP on intracranial pressure. The primary outcome variable was the change in volume / area of the choroid (mean±sd). Lying in the -6° head down tilt position for three days without the use of a pillow caused an increase the choroid area (Δ0.11 mm2, p=0.05) and volume (Δ0.45 mm3, p=0.003). If participants spent 8 hours per day under low-level LBNP, the choroid still increased in volume, but substantially (40%) less than in the control trial (Δ0.27 mm3, p=0.05). Moreover, the increase in choroid area was completely abolished (Δ0.03 mm2, p=0.13). LBNP caused a reduction in intracranial pressure, which remained below the -6° head down tilt value for 8 hours while the device was on and returned to normal after the device was switched off (-6 deg, 8am, 14.5±5.0; 8 hrs LBNP, 11.6±3.8; 8pm 13.6±4.4mmHg). Eight hours per day of low-level LBNP substantially attenuates the choroid expansion associated with 3 days of strict -6 deg head down tilt bedrest. These data provide evidence that low-level LBNP maybe an effective countermeasure for Space flight-associated neuro-ocular syndrome.