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This study was performed to investigate the interocular symmetry of peripapillary retinal nerve fibre layer (RNFL) thickness, as measured by Cirrus high-definition spectral-domain optical coherence tomography (OCT), in healthy eyes. A wide range of subject ages and refractive errors was examined.The retinal nerve fibre layer thickness was measured in 1,234 healthy eyes from 617 subjects using OCT. Interocular differences (right eye minus left eye) in global area and quadrant nerve fibre layer thicknesses were measured. The effect of age and refractive error on interocular nerve fibre layer thickness difference was also examined.Means (and standard deviations) of subjects' ages and average subject refractive errors were 36.4 ± 19.8 years (range: five to 80 years) and -2.67 ± 2.91 D (range: -14.13 to +5.75 D), respectively. Cutoff limits for normal interocular nerve fibre layer thickness differences (2.5th and 97.5th percentiles of normative data) in the global area and in the superior, nasal, inferior and temporal quadrants were 9.5, 23.0, 15.6, 20.0 and 22.6 μm, respectively. Mean interocular retinal nerve fibre layer thickness differences in global area and in superior, nasal, inferior and temporal quadrants were 0.7 (p < 0.001), -3.9 (p < 0.001), 2.6 (p < 0.001), 1.1 (p = 0.007) and 3.0 (p < 0.001) μm, respectively. Interocular nerve fibre layer thickness differences were not significantly correlated with age or refractive error (average of right and left eyes, both p > 0.05).Significant interocular differences in peripapillary retinal nerve fibre layer thickness exist in healthy eyes, particularly in the superior quadrant. This finding should be considered when comparing retinal nerve fibre layer thickness between right and left eyes.