Purpose-To examine the impact of pupil center location uncertainty in wavefront sensing on the variance in repeated measures of the high order RMS wavefront error (WFE).Methods-Dilated WFE for one normal eye and one eye with keratoconus were measured using a custom Shack/Hartmann wavefront sensor (lenslet spacing 400 μm). Twelve measurements for each subject were averaged to form the best estimate of each eye's WFE and standard deviation. The percentage of the standard deviation of the actual measurements attributable to the uncertainty in pupil center location was modeled by inducing random offsets in the pupil center up to 50, 100, 150 and 200 μm.Results-The percentage of standard deviation of the actual measurements accounted for by pupil location uncertainty form a complex interaction between magnitude and distribution of the HO-WFE with the magnitude of pupil centering error. The larger the WFE and the larger the pupil center uncertainty the greater is the effect.Conclusions-As pupil center uncertainty increases, so does the WFE variation in repeated measurements. The larger the underlying WFE, the greater is the impact on measurement variation. Increasing measurement variation decreases the ability to detect changes in WFE (e.g., as a function of aging or clinical intervention) and decreases the accuracy with which WF guided corrections can be made.