Evaluate the effect of repeated doses of topical 1% cyclopentolate hydrochloride alone and in combination with topical 2.5% phenylephrine on pupil diameter (PD), tear production (STT-1), intraocular pressure (IOP), digestive function (gut motility and feces production), and heart rate (HR). Six healthy mares. In a prospective, randomized, controlled, and crossover design study, the left eye of six healthy mares was administered 0.2mL of cyclopentolate alone and in combination with 0.2mL of phenylephrine. The drugs were administered 3 times a day for 1day, twice a day for 1day, and then once a day for 2days, as commonly used in practice. Daily and two days after the last topical drug administration, HR, digestive auscultation, feces production, STT-1, IOP, and PD were recorded. The cyclopentolate alone significantly increased the horizontal and vertical PD of the treated eye from day 2 to day 6 (p<.0001) compared with the baseline value. The combination with topical phenylephrine did not have any additional effect on mydriasis compare with the cyclopentolate alone. The other ocular and digestive parameters were not affected by repeated doses of cyclopentolate alone or combined. Repeated administration of cyclopentolate alone or combined with phenylephrine induce a significant mydriasis for at least 48h after the last administration in normal horses' eyes, and do not affect STT-1, IOP, digestive function, and HR. The phenylephrine combined with the cyclopentolate did not potentiate the pupil dilation when compared with cyclopentolate alone in healthy horses.