PurposeTo evaluate the measurement of anisocoria in a group of ocular healthy subjects using a standardized protocol in scotopic, mesopic, and photopic lighting conditions, and determine the optimal threshold of difference in pupil diameter in determining physiologic anisocoria.MethodsRight and left pupil diameters of 126 ocular healthy subjects with a mean age 30.5±7.8 years (40 males and 86 females) were measured sequentially under photopic conditions using a monocular infrared pupillometer. A sub-group of 51 individuals had right and left pupil measurements performed under three additional lighting conditions, allowing for a 2-minute recovery between measurements. A white light emitting diode (LED) in the eyecup of the pupillometer produced three controlled light settings: scotopic (0 lux), low mesopic (0.3 lux), and high mesopic (3 lux). The criterion for anisocoria was defined as ≥0.4 mm difference in pupil diameter between the eyes.ResultsIn the 126 subjects tested, 23.8% (n=30) exhibited anisocoria in photopic conditions. In the sub-group measured under three additional light settings, 43.1% (n=22) exhibited anisocoria in scotopic conditions, 43.1% (n=22) in low mesopic conditions, and 47.1% (n=24) in high mesopic conditions. Approximately 73% of subjects exhibited anisocoria in at least one light setting, while only approximately 8% had anisocoria in every light setting. When the criterion for anisocoria was shifted to ≥0.2 mm or ≥0.6 mm, the prevalence of anisocoria shifted significantly. Using a higher cutoff of ≥0.6 mm effectively reduced the number of healthy individuals who exhibit anisocoria in every light setting to almost zero.ConclusionBased on our data, anisocoria is more prevalent under varied lighting conditions. To ensure the anisocoria is due to physiologic reasons, one should ensure that it is present under all lighting conditions to avoid excessive false positives.