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To evaluate the repeatability and between-methods differences of optic nerve head and retinal nerve fiber layer thickness (RNFLT) measurements as made with the automatic, operator-adjusted, and manual disc-definition functions of the RTVue-100 Fourier-domain optical coherence tomograph.One eye of each of 126 persons (72 glaucomatous, 10 ocular hypertensive, 44 healthy control eyes) was analyzed with all 3 disc-definition functions. Thirty-three eyes were imaged 5 times for the determination of measurement repeatability.For several parameters, repeatability with automatic disc definition was significantly poorer than the other options. In contrast, for all but 1 parameter repeatability did not differ between the manual and operator-adjusted methods. Refractive error, disease category or peripapillary atrophy had no influence on repeatability. However, the measured values differed significantly between the methods, for all but 3 parameters. Similar between-method differences were seen for eyes irrespective of glaucoma, refractive error, or parapapillary atrophy. High intraclass correlation (93.5% to 99.9%) was seen for all parameters measured with manual and operator-adjusted methods, but in the other comparisons only for RNFLT parameters. Agreement of the classifications was better for RNFLT parameters than disc parameters, and the κ-values were highest (0.881 to 0.968) in comparing the manual and operator-adjusted methods.Measurement repeatability with manual and operator-adjusted disc-definition functions is similar and is not influenced by refractive error, glaucoma, or parapapillary atrophy. The high intraclass correlation and the agreement of diagnostic classifications suggest that operator-adjusted disc-definition can be useful for glaucoma detection with the RTVue-100 Fourier-domain optical coherence tomograph.