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Background: First reported study to assess the effect of diurnal variation on anterior chamber angle measurements, as well as, to re-test the effects of lighting and angle-of-incidence variation on anterior chamber angle (ACA) measurements acquired by time-domain anterior segment optical coherence tomography (AS-OCT).Methods: A total of 30 eyes from 15 healthy, normal subjects underwent anterior chamber imaging using a Visante time-domain AS-OCT according to an IRB-approved protocol. For each eye, the inferior angle was imaged twice in the morning (8 am - 10 am) and then again in the afternoon (3 pm - 5 pm), under light meter-controlled conditions with ambient room lighting 'ON' and lights 'OFF', and at 5° angle of incidence increments. The ACA metrics measured for each eye were: angle opening distance (AOD, measured 500 and 750 μm anterior from scleral spur), the trabecular-iris-space area (TISA, measured 500 and 750 μm anterior from scleral spur), and scleral spur angle. Measurements were performed by masked, certified Reading Center graders using the Visante's Internal Measurement Tool. Differences in measurements between morning and afternoon, lighting variations, and angle of incidence were compared.Results: Mean age of the participants was 31.2 years (range 23-58). Anterior chamber angle metrics did not differ significantly from morning to afternoon imaging, or when the angle of incidence was offset by 5° in either direction away from the inferior angle 6 o'clock position. (p-value 0.13-0.93). Angle metrics at the inferior corneal limbus, 6 o'clock position (IC270), with room lighting 'OFF', showed a significant decrease (p < 0.05) compared to room lighting 'ON'.Conclusions: There does not appear to be significant diurnal variation in AS-OCT parameters in normal individuals, but lighting conditions need to be strictly controlled since variation in lighting led to significant variability in AS-OCT parameters. No changes in ACA parameters were noted by varying the angle-of-incidence, which gives confidence in being able to perform longitudinal studies in approximately the same area (plus/minus 5° of original scan location). [ABSTRACT FROM AUTHOR]