Intracranial pressure increases in head-down tilt (HDT) body posture. This study evaluated the effect of HDT on the optic nerve sheath diameter (ONSD) in normal subjects. Twenty six healthy adults (age 28 [4.7] years) participated in seated and 6° HDT visits. For each visit, subjects presented at 11:00 h for baseline seated scans and then maintained a seated or 6° HDT posture from 12:00 to 15:00 h. Three horizontal axial and three vertical axial scans were obtained at 11:00, 12:00 and 15:00 h with a 10 MHz ultrasonography probe on the same eye, randomly chosen per subject. At each time point, horizontal and vertical ONSD (mm) were quantified by averaging three measures taken 3 mm behind the globe. In the seated visit, ONSDs were similar across time (p > 0.05), with an overall mean (standard deviation) of 4.71 (0.48) horizontally and 5.08 (0.44) vertically. ONSD was larger vertically than horizontally at each time point (p < 0.001). In the HDT visit, ONSD was significantly enlarged from baseline at 12:00 and 15:00 h (p < 0.001 horizontal and p < 0.05 vertical). Mean (standard error) horizontal ONSD change from baseline was 0.37 (0.07) HDT versus 0.10 (0.05) seated at 12:00 h (p = 0.002) and 0.41 (0.09) HDT versus 0.12 (0.06) seated at 15:00 h (p = 0.002); mean vertical ONSD change was 0.14 (0.07) HDT versus -0.07 (0.04) seated at 12:00 h (p = 0.02) and 0.19 (0.06) HDT versus -0.03 (0.04) seated at 15:00 h (p = 0.01). ONSD change in HDT was similar between 12:00 and 15:00 h (p ≥ 0.30). Changes at 12:00 h correlated with those at 15:00 h for horizontal (r = 0.78, p < 0.001) and vertical ONSD (r = 0.73, p < 0.001). The ONSD increased when body posture transitioned from seated to HDT position without any further change at the end of the 3 h in HDT.