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This study examined the effects of ophthalmic timolol and time of administration on cardiovascular and respiratory functions in healthy young male volunteers. Eight participants (mean age ± standard deviation, 22 ± 0.9 years) received either 50 µL of 0.5% timolol or placebo in the lower conjunctival sacs of both eyes in the morning or evening. Intraocular pressure, blood pressure, heart rate, and respiratory functions, including percent forced expiratory volume in 1 second and peak expiratory flow rate, were then measured for 3 hours after drug administration. Timolol reduced intraocular pressure and cardiovascular function at both administration times. However, a timolol-induced reduction in respiratory function was observed only in the evening: percent forced expiratory volume in 1 second, peak expiratory flow rate, and expiratory flow rate at 75% vital capacity were reduced by 3%, 7%, and 12%, respectively, 3 hours after administration. These results indicate that ophthalmic timolol reduces cardiovascular and respiratory functions in healthy young male subjects and that bronchial sensitivity to timolol differs between morning and evening.