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To develop a measurement protocol for changes in the shape and size of the ciliary muscle with accommodation using the Zeiss Visante™ anterior segment optical coherence tomography (AS-OCT) and to determine the test-retest repeatability of these measurements.Subjects were 25 adults aged 23 to 28 years. The ciliary muscle was imaged at two visits with the Visante™ while accommodative response was monitored during imaging using the PowerRefractor. Ciliary muscle thickness (CMT) was measured at 1 mm (CMT1), 2 mm (CMT2), and 3 mm (CMT3) posterior to the scleral spur and at the point of maximal thickness (CMTMAX). Thickness was measured at these locations while subjects viewed a target at distance and at a 4.00 D accommodative stimulus. Outcome measures were the change in thickness between distance and the 4.00 D stimulus and the change in thickness per diopter of accommodative response (PowerRefractor). Finally, the repeatability measurements between visit 1 and visit 2 were determined with a Bland-Altman analysis.The statistically significant modeled changes in CMT were as follows: CMTMAX = 69.2 μm (4.00 D stimulus) and 18.1 μm (per diopter of accommodation); CMT1 = 45.2 μm (4.00 D stimulus) and 12.3 μm (per diopter of accommodation); and CMT3 = −45.9 μm (4.00 D stimulus) and −12.0 μm (per diopter of accommodation); p < 0.0001 for all.The combination of the Visante™ and the PowerRefractor is a feasible tool for measuring thickening of ciliary muscle at more anterior locations and thinning at more posterior locations during accommodation. We noted a wide range of accommodative responses during the time of image capture in this study indicating that the most accurate estimates of the change in ciliary muscle dimensions with accommodation may be obtained by using accommodative response rather than stimulus values and by using measurements taken simultaneously with image capture.