PURPOSE: To evaluate repeatability and positional independence of optic nerve head (ONH) and retinal nerve fiber layer (RNFL) thickness measurements in sitting and supine body positions using portable spectral-domain optical coherence tomography (iVue SD-OCT; Optovue Inc). DESIGN: Evaluation of diagnostic technology. METHODS: Sixty eyes of 30 subjects (10 healthy younger adults aged 20-27 years, 10 healthy older adults aged 50-66 years, and 10 glaucoma patients aged 38-82 years) were included prospectively. For each participant, all measurements were taken in a single session. After 5 minutes in the supine position, 5 scans were obtained from both eyes. Following a 5-minute sitting adaptation, 5 scans were then obtained in the sitting position. The same instrument was used for all measurements. Repeatability and correlation between supine and sitting measurements of 4 ONH and 3 RNFL parameters were assessed using intraclass correlation coefficients (ICC), concordance correlation coefficients (ρ), and Bland-Altman plots. RESULTS: Measurements were highly repeatable within individual eyes, both for ONH (ICC range, 73%-99%) and RNFL (ICC range, 72%-99%) parameters. The correlation between supine and sitting ONH measurements was strong and ranged from ρ = 97%-99% (younger healthy) to ρ = 98%-99% (older healthy) and ρ = 84%-99% (glaucoma). Bland-Altman plots indicated good agreement between sitting and supine readings of ONH and RNFL parameters. CONCLUSIONS: Repeatability of measurements of ONH and RNFL is high and measurements between sitting and supine are highly correlated. The ability of the iVue SD-OCT to evaluate ONH and RNFL parameters is good to excellent in both body positions.