OBJECTIVE To describe the pattern and magnitude of diurnal variation of choroidal thickness (CT), its relation to systemic and ocular factors, and to determine the intervisit reproducibility of diurnal patterns. METHODS A prospective study was conducted on 12 healthy volunteers who each underwent sequential ocular imaging on two separate days at five fixed, 2-hour time intervals. Spectral domain optical coherence tomography (OCT) with enhanced depth imaging and image tracking was performed using a standardized protocol. Choroidal and retinal thicknesses were independently assessed by two masked graders. CT diurnal variation was assessed using repeated-measures ANOVA. RESULTS A significant diurnal variation in CT was observed, with mean maximum CT of 372.2 μm, minimum of 340.6 μm (P < 0.001), and mean diurnal amplitude of 33.7 μm. Retinal thickness (mean, 235.0 μm) did not exhibit significant diurnal variation (P = 0.621). The amplitude of CT variation was significantly greater for subjects with thicker morning baseline CT compared with those with thin choroids (43.1 vs. 10.5 μm, P < 0.001). There were significant correlations between amplitude of CT and age (P = 0.032), axial length (P < 0.001), and spherical equivalent (P < 0.001). The change in CT also correlated with change in systolic blood pressure (P = 0.031). Comparing CT on two different days, a similar diurnal pattern was observed, with no significant difference between corresponding measurements at the same time points (P = 0.180). CONCLUSIONS There is significant diurnal variation of CT, with good intervisit reproducibility of diurnal patterns on two different days. The amplitude of variation varies with morning baseline CT, and is correlated with age, axial length, refractive error, and change in systolic blood pressure.