PURPOSE: To determine whether letter sequences and/or lens-presentation order should be randomized when measuring defocus curves and to assess the most appropriate criterion for calculating the subjective amplitude of accommodation (AoA) from defocus curves. SETTING: Eye Clinic, School of Life & Health Sciences, Aston University, Birmingham, United Kingdom. METHODS: Defocus curves (from +3.00 diopters [D] to -3.00 D in 0.50 D steps) for 6 possible combinations of randomized or nonrandomized letter sequences and/or lens-presentation order were measured in a random order in 20 presbyopic subjects. Subjective AoA was calculated from the defocus curves by curve fitting using various published criteria, and each was correlated to subjective push-up AoA. Objective AoA was measured for comparison of blur tolerance and pupil size. RESULTS: Randomization of lens-presentation order and/or letter sequences, or lack of, did not affect the measured defocus curves (P>.05, analysis of variance). The range of defocus that maintains highest achievable visual acuity (allowing for variability of repeated measurement) was better correlated to (r = 0.84) and agreed best with ( 0.50 D) subjective push-up AoA than any other relative or absolute acuity criterion used in previous studies. CONCLUSIONS: Nonrandomized letters and lens presentation on their own did not affect subjective AoA measured by defocus curves, although their combination should be avoided. Quantification of subjective AoA from defocus curves should be standardized to the range of defocus that maintains the best achievable visual acuity.