Conjunctival microcirculation imaging provides a non-invasive means for detecting hemodynamic alterations due to systemic and ocular diseases. However, reliable longitudinal monitoring of hemodynamic changes due to disease progression requires establishment of measurement variability over time. The purpose of the current study was to determine inter-visit variability of conjunctival microvascular hemodynamic measurements in non-diabetic control (NC, N = 7) and diabetic retinopathy (DR, N = 10) subjects. Conjunctival microvascular imaging was performed during 2 visits, which were 17 ± 12 weeks apart. Images were analyzed to determine vessel diameter (D), axial blood velocity (V), blood flow (Q), wall shear rate (WSR) and wall shear stress (WSS). The inter-visit variability was determined based on mean inter-visit differences. In NC, inter-visit variability of D, V, Q, WSR and WSS were 0.2 ± 0.5 μm, −0.01 ± 0.16 mm/s, −8 ± 46 pl/s, −3 ± 46 s−1 and −0.01 ± 0.10 dyne/cm2, respectively. Inter-visit variability of D, V, Q, WSR and WSS were beyond the normal 95% confidence limits in 60%, 20%, 40%, 20% and 20% of DR subjects, respectively. The variability of hemodynamic measurements over time was established in non-diabetic subjects, suggestive of the potential of the method for detecting longitudinal changes due to progression of DR.