To evaluate the effect of bimatoprost/timolol maleate fixed combination (BTFC), latanoprost/timolol maleate fixed combination (LTFC), and travoprost/timolol maleate fixed combination (TTFC) on 24-h intraocular pressure (IOP) in patients with open-angle glaucoma. This prospective, observer-masked, randomized study included 50 patients with primary open-angle glaucoma. All patients were using hypotensive lipids and timolol maleate fixed combination treatment for ≥4weeks and had an IOP≤21mmHg. Group 1 (n=18) received BTFC, group 2 (n=14) received LTFC, and group 3 (n=18) received TTFC. All patients were hospitalized, and IOP was monitored for 24-h (10:00, 14:00, 18:00, 22:00, 02:00, and 06:00). Mean diurnal IOP variation measurements were taken between 06:00 and 18:00, and mean nocturnal IOP variation measurements were taken between 22:00 and 02:00. Mean IOP and IOP variation in the three groups were compared. Mean 24-h IOP did not differ significantly between the three groups (group 1: 14.6±2.9mmHg; group 2: 14.1±3.7mmHg and group 3: 15.8±2.0mmHg; P>0.05). Mean diurnal IOP variation was 4.6±2.3mmHg in group 1, 5.8±2.4mmHg in group 2, and 4.3±1.7mmHg in group 3, and mean nocturnal IOP variation was 3.2±2.8mmHg in group 1, 2.9±1.9mmHg in group 2, and 3.0±1.6mmHg group 3. There were not any significant differences in diurnal or nocturnal IOP variation between the three groups (P<0.05). All three fixed combinations effectively controlled IOP for 24-h and had a similar effect on diurnal and nocturnal IOP variations.