To assess the reliability and validity of spectral-domain optical coherence tomography (SD-OCT) measurements of retinal vessel lumen diameters and wall thicknesses. SD-OCT was used to characterize the circular region around the optic disc of 40 eyes (20 subjects). The inner and outer sides (vitreal and choroidal sides) of the vessel wall and the luminal diameter were measured using intensity graphs. Mean arterial and venous luminal diameters were 95.1±16.1 and 132.6±17.8 μm, respectively. The wall thicknesses of inner and outer sides of the artery were 23.9±4.9 and 21.2±3.5 μm, respectively. The wall thicknesses of the inner and outer sides of the vein were 20.7±4.2 and 16.3±4.3 μm, respectively. There were significant differences between the inner and outer wall thicknesses in both the artery and vein (P<0.01). Intra- and interobserver intraclass correlation coefficients (ICCs) for lumen measurements were >0.95, and for wall thicknesses were >0.85, except for the outer wall thickness measurements. The mean value of outer and inner wall thicknesses showed good reproducibility, with ICCs of >0.85. Intensity graph-assisted measurements using SD-OCT provided more objective information in finding boundaries of vessels. Luminal diameters and wall thicknesses obtained with OCT showed good overall reproducibility, with inner wall thicknesses being thicker, and with better reproducibility compared with outer wall thicknesses, where ICC values were the lowest among the inner wall thicknesses, mean thicknesses of inner and outer walls, and luminal diameters. When using SD-OCT measurements, caution is therefore advised when using only the outer wall as representative of the wall thicknesses.