The application of spectral domain optical coherence tomography as a surrogate for neurodegeneration in a range of neurological disorders demands better understanding of the physiological variation of retinal layer thicknesses, which may mask any value of this emerging outcome measure. A prospective study compared retinal layer thicknesses between control subjects (n = 15) and runners (n = 27) participating in a 10-km charity run. Three scans were performed using an eye-tracking function (EBF) and automated scan registration for optimal precision at (1) baseline, (2) directly after the run, and (3) following a rehydration period. Retinal layer segmentation was performed with suppression of axial retinal vessel signal artifacts. Following the run, there was an increase in the relative retinal nerve fibre layer (p = 0.018), the combined inner plexiform/ganglion cell layer (p = 0.038), and the outer nuclear layer (p = 0.018) in runners compared to controls. The initial increase of thickness in the outer nuclear layer of runners (p