With the growing availability of high-resolution imaging there has been increased interest in developing new metrics for integrity of the retinal nerve fiber layer. In particular, it has been suggested that measurement of width of retinal nerve fiber bundles (RNFBs) may be useful in glaucoma, due to low between-subject variability in mean RNFB width. However, there have also been reports of substantial within-subject variability in the width of individual RNFBs. To assess within-subject variability as a potential source of selection bias in measurements of RNFB width, we used an adaptive optics scanning laser ophthalmoscope (AOSLO) to measure widths of individual RNFBs in one eye each of 11 young adults in good ocular health. In a pilot study we analyzed a large AOSLO image of RNFL in one participant then, based on those findings, in the main study we used AOSLO to image a smaller region in 10 additional healthy young adults. The pilot study of one eye found RNFB widths ranging from 10 μm to 44 μm. This suggested that biological variability was too high for measuring small changes arising from disease processes. This was confirmed in measurements of 10 eyes in the main study, RNFB widths ranged from 9 μm to 55 μm and every eye had large within-subject variability (exceeding 19 μm in all eyes) in RNFB width for nearby bundles. The within-subject variability in RNFB width, as well as variation in the width of single RNFBs over relatively short distances (<300 um) depending on the precise location of measurement, suggests that bundle width measurements would be highly susceptible to selection bias and therefore of limited clinical use.