Purpose To prospectively evaluate the diurnal variation of intraocular pressure (IOP) during unoprostone treatment in 13 healthy volunteers. Method IOP was measured by Goldmann applanation tonometry by the same observer every 3 hours from 9 am to 9 am the next morning. Unoprostone was then instilled at 9 am and 9 pm daily for 1 month. After 1 month, IOP was measured again with unoprostone instilled at 9 am and 9 pm during IOP measurement. We then compared the average daily IOP before and after the treatment (paired t-test). Results After 1 month of treatment, the average IOP decreased at every time point but one (12 pm, 3 pm, 6 pm, 9 pm, 12 am, 3 am, and 9 am, but not at 6 am). There were no adverse reactions and none of the subjects discontinued unoprostone. Conclusion The hypotensive effects of unoprostone persist throughout the day, but this study suggests that the effects may be weaker at nighttime and early in the morning.