This study was designed to evaluate the effect of microgravity on the diurnal variation of intraocular pressure (IOP). IOPs were measured with the pressure phosphene tonometer in 1 subject (the first Korean astronaut) during spaceflight. IOPs were measured every 3 hours during day time (6 times per day) at 2 separate days in space with 3 repeated measurements at each time on both eyes. A total of 72 measurements were obtained during spaceflight. To obtain control IOP data, IOP was measured using the same protocol on ground before spaceflight. Mean IOP increased by 26.3% during spaceflight compared with that on ground [16.47 ± 0.60 (SD) mm Hg vs. 13.04 ± 0.74 mm Hg, P<0.001). The IOP elevation was maintained until Launch+8 days. There was no significant difference in IOP increase between right and left eyes (16.4 2 ± 0.65 mm Hg right eye vs. 16.53 ± 0.56 mm Hg left eye). There was a different pattern of diurnal variation of IOP during spaceflight compared with that on ground. The IOP at 7 AM was the lowest under microgravity, whereas it was the highest on ground. The slope of the best fit line for diurnal IOP measures was 0.0349 mm Hg/h (95% confidence interval: 0.0082-0.0616) under microgravity and -0.0294 mm Hg/h (95% confidence interval: -0.0063-0.0041) on ground. The study showed a different diurnal pattern of IOP under microgravity compared with that on ground. This result suggests that gravity and subsequent body fluid shift is one of the determining factors of IOP diurnal variation.