PURPOSE: To determine the effect of optical defocus on the test-retest variability (TRV) of visual acuity measurements in normal subjects. METHODS: Normal subjects underwent repeated visual acuity measurement with optical defocus of 0, 0.50, and 1.00 D. All measurements were taken using the Early Treatment Diabetic Retinopathy Study (ETDRS) version of the Bailey-Lovie logMAR chart. TRV was quantified in terms of its 95% range, both empirically and using the approach of Bland and Altman. RESULTS: According to the Bland and Altman approach, the estimated 95% TRV ranges were +/-0.11 logarithm of the minimum angle of resolution (logMAR) for 0-D defocus, +/-0.18 logMAR for 0.50-D defocus, and +/-0.25 logMAR for 1.00-D defocus. CONCLUSIONS: Optical defocus has a considerable effect on the TRV of visual acuity measurements. These findings have important implications for both clinical practice and clinical research. Uncorrected refractive errors as small as 0.50 D may compromise the detection of visual change in individuals, and contribute to unnecessarily large sample sizes in clinical trials in which visual acuity is used as a primary outcome measure.