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More than half of long-duration spaceflight astronauts have experienced ophthalmic abnormalities, collectively termed spaceflight-associated neuro-ocular syndrome (SANS). Microgravity-induced elevation in intracranial pressure(ICP), especially optic nerve cerebral spinal fluid pressure (ON-CSFp) due to headward fluid shifts is the primary hypothesized contributor to SANS. However there is limited non-invasive method to monitor ON-CSFp. The present longitudinal study aims to quantify the area of the optic nerve subarachnoid space(ONSSA) in the axial plane behind the optic disc, that is filled with CSF and expands when ON-CSFp is elevated, during and after head-down tilt (HDT) bed rest (BR) and assess its association with relative anthropometric and systemic parameters.72 eyes of 36 healthy male volunteers were included and underwent 90-day strict 6° HDT BR .The measurements were performed on day 30, 60, and 90 of HDT BR and at six recovery time points extending to 180-day in supine position. Portable B-scan ultrasound was performed binocularly, using a 12 MHz linear array probe. The measurement of the optic nerve sheath and calculation of the ONSSA from 3mm to 5mm posterior to the optic disc were performed with ImageJ 1.51 analysis software by two experienced observers in a masked manner. In comparison to recovery day 180, ONSSA from 3mm to 5mm posterior to the optic disc exhibited significant distention on BR day 30,60 and 90, with a mean change of 0.4mm2 (95% CI:0.12 to 0.7 mm2, P = .002), 0.35 mm2 (95% CI:0.02 to 0.69mm2, P = .001), and 0.4 mm2 (95% CI:0.05 to 0.75 mm2, P = .015), respectively. Univariate linear regression analysis showed increased ONSSA was correlated with increased mean blood pressure (Beta = 0.092; 95%CI, 0.016 to 0.169; P = .019). Terrestrially, ONSSA distension is associated with increased ICP. The results indicated that 90-day HDT BR induced elevated ICP especially ON-CSFp in comparison to supine level.Advanced quantitative portable ultrasound-based assessment of the ONSSA could better our understanding the role of ON-CSFp on SANS.