Objective: To determine whether the order of intraocular pressure (IOP) measurement between right and left eyes affects IOP measurement. Methods: A total of 105 healthy volunteers from the eye clinics and staff at Washington University were randomized into 2 groups. Group 1 underwent 3 sets of 2 IOP measurements per eye, starting with the right eye (right eye twice, left eye twice, right eye twice). Group 2 underwent similar measurements starting with the left eye. After 2 weeks the order of IOP measurements were reversed between groups. A mixed-model repeated-measures analysis of variance analyzed the association of IOP measurement with the order measured (1) between first and second eyes, (2) between first and second visits, (3) between right and left eyes, and (4) with ocular squeezing. Results: Intraocular pressure measured higher in first eyes compared with fellow eyes regardless of whether right or left eyes were measured first (P=.002). Intraocular pressure measurements decreased between the first and second visits (P<.001). No difference was found in IOP measurements between right and left eyes (P=.41). Moderate and severe ocular squeezing were associated with higher IOP measurements (P<.001) and occurred more during earlier than later IOP measurements within a set (P<.001) and between sets (P<.001 to P=.03). Conclusions: Intraocular pressure measured in the first eye, whether right or left, is higher than IOP measured in the fellow eye; this may be partially because of ocular squeezing. Measurements of IOP decrease between first and second visits. Multiple IOP measurements at multiple visits are necessary to accurately diagnose and treat patients with glaucoma and ocular hypertension.