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The aim of the present study was to investigate the reproducibility and potential diurnal variation of optic nerve head and retinal blood flow parameters in healthy individuals over a period of 12 hr.We measured optic nerve head and retinal blood flow parameters in 16 healthy male non-smoking individuals at five time-points during the day (08:00, 11:00, 14:00, 17:00 and 20:00 hr). Outcome parameters were perimacular white blood cell flux (as assessed with the blue field entoptic technique), blood velocities in retinal veins (as assessed with bi-directional laser Doppler velocimetry), retinal arterial and venous diameters (as assessed with the retinal vessel analyser), optic nerve head blood flow, volume and velocity (as assessed with single point and scanning laser Doppler flowmetry) and blood velocities in the central retinal artery (as assessed with colour Doppler imaging). The coefficient of variation and the maximum change from baseline in an individual were calculated for each outcome parameter.No diurnal variation in optic nerve head or retinal blood flow was observed with any of the techniques employed. Coefficients of variation were between 1.6% and 18.5% for all outcome parameters. The maximum change from baseline in an individual was much higher, ranging from 3.7% to 78.2%.Our data indicate that in healthy individuals the selected techniques provide adequate reproducibility to be used in clinical studies. However, in patients with eye diseases and reduced vision the reproducibility may be considerably worse.