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OBJECTIVE To determine effects of diurnal variation and anesthetic agents on intraocular pressure (IOP) in Syrian hamsters (Mesocricetus auratus). ANIMALS 90 healthy adult Syrian hamsters (45 males and 45 females). PROCEDURES IOP was measured with a rebound tonometer. In phase 1, IOP was measured in all hamsters 3 times during a 24-hour period (7 am, 3 pm, and 11 pm). In phase 2, hamsters were assigned to 5 groups (18 animals [9 males and 9 females]/group). Each group received an anesthetic agent or combination of anesthetic agents (ketamine hydrochloride, xylazine hydrochloride, diazepam, ketamine-diazepam [KD], or ketamine-xylazine [KX] groups) administered via the IP route. The IOP was measured before (time 0 [baseline]) and 10, 30, 60, 90, 120, and 150 minutes after administration of drugs. RESULTS Mean ± SD IOP values were 2.58 ± 0.87 mm Hg, 4.46 ± 1.58 mm Hg, and 5.96 ± 1.23 mm Hg at 7 am, 3 pm, and 11 pm, respectively. Mean baseline IOP was 6.25 ± 0.28 mm Hg, 6.12 ± 0.23 mm Hg, 5.75 ± 0.64 mm Hg, 5.12 ± 1.40 mm Hg, and 4.50 ± 1.30 mm Hg for the ketamine, xylazine, diazepam, KD, and KX groups, respectively. A significant decrease in IOP, compared with baseline IOP, was detected in only the KX group at 30, 60, and 90 minutes after drug administration. CONCLUSIONS AND CLINICAL RELEVANCE Maximum IOP in Syrian hamsters was detected at night. The ketamine-xylazine anesthetic combination significantly decreased IOP in Syrian hamsters.