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Purpose: This study investigates the influence of age and axial length (AL) on the peripapillary retinal nerve fiber layer (RNFL) thickness, as measured by optical coherence tomography (OCT). Methods: Healthy patients visiting an eye clinic at a county hospital were recruited. All participants underwent comprehensive ophthalmologic examinations, and their retinas were scanned using 3D OCT-1000. In total, 223 patients with 446 eyes were included. The mean age and AL were 42.07 ± 13.16 (21–76) years and 25.38 ± 1.73 (21.19–30.37) mm, respectively. Results: The average RNFL thickness decreased by 2.71 μm for every 10-year increase in age (P < 0.001). Age-related RNFL thinning was more significant in participants older than 41 years (-0.24μm/year; P = 0.015). The earliest sector showing a significant decline in RNFL thickness was after 35 years of age (-0.70μm/year; P = 0.011) at the superior quadrant and at the 1–2 o’clock hour (-1.42μm/year; P = 0.009). Meanwhile, the maximal rate of age-associated RNFL decay was observed in these two regions as well. The reduction of RNFL with age progression did not differ in eyes with long AL (> 27 mm; -0.16μm/year) or those with short AL (< 25 mm; -0.22μm/year). For every 1-mm-greater AL, RNFL was thinner by 1.78 μm (P < 0.001). The inferior quadrant showed the greatest tendency of RNFL decline with longer AL (4.46 μm/mm; P < 0.001). Conclusions: The factors of age and AL should be considered when interpreting the results. Significantly age-associated RNFL thinning was found in participants older than 41 years. Reduction of RNFL thickness with increasing age was not affected by AL. Topographic variations in RNFL thinning were observed in that the maximal decline of RNFL thickness with advancing age at the superior quadrant whereas with elongation of AL at the inferior quadrant. [ABSTRACT FROM AUTHOR]