SIGNIFICANCE:This is the first study to validate the repeatability of objective measurements of amplitude of accommodation (AA) using the TONOREF III (NIDEK Co., Ltd., Japan), which can measure the AA in 30 seconds. PURPOSE:To evaluate the repeatability of objective measurements of AA using the TONOREF III and explored the association between objectively measured AA and factors, including age, sex, spherical equivalent, baseline pupil size and pupil size change during accommodation. METHODS:This cross-sectional study recruited 35 healthy subjects aged 26 to 52 years. The Bland-Altman method and intraclass correlation coefficients (ICCs) were used to assess the repeatability of TONOREF III measurements. The relationships between AA and age, sex, baseline pupil size, changes in pupil size (ΔPS) during accommodation were analyzed using linear regression for univariate and multivariate analysis. RESULTS:The mean difference in AA (ΔAA) between two sessions of TONOREF III measurements was 0.23 D (95% CI: -1.07 to +1.53 D), while no significant correlation was found between the mean and ΔAA (p = .14). The ICCs of the TONOREF III was 0.96. Age, sex, and ΔPS during accommodation were significant factors affecting the AA in multivariate analysis. CONCLUSIONS:The repeatability of objective AA measurements using the TONOREF III was good. Measuring AA using the TONOREFTM III in clinical practice is feasible.