PURPOSE. With the introduction of spectral domain-optical coherence tomography (SD-OCT), changes in retinal nerve fiber layer (RNFL) thickness and macular volume (MV) can be detected with high precision. The aim of this study was to determine whether there is a physiological quantifiable degree of variation of these structures in humans. METHODS. This study took place during a 10-km charity run at VU University Medical Center Amsterdam. Weight, height, hydration status, RNFL thickness (ring scan, 12° around the optic nerve head), and MV (20° × 20°) were assessed in 69 subjects (44 runners, 25 controls) using SD-OCT with eye-tracking function. The SD-OCT scans were assessed before running (normal status), after running (more dehydrated status), and 1 to 1.5 hours after finishing the run (rehydrated status). Controls were measured at the same time intervals as the runners but did not participate in the running event. Changes over time were assessed by general linear models, correcting for repeated measurements. RESULTS. In runners, a significant increase in both RNFL thickness (94.4 μm [baseline] to 95.2 μm [rehydration], P = 0.04) and MV (288.9 μm [baseline] to 291.0 μm [rehydration], P < 0.001) over time was observed. Controls did not show significant changes over time. Anatomically, the physiological change of RNFL thickness was most marked in the nasal sectors. CONCLUSIONS. This prospective study demonstrated a significant physiological variation of the RNFL thickness and MV at a proportion that, on an individual patient level, may be relevant for longitudinal studies in neurodegenerative diseases. © 2012 The Association for Research in Vision and Ophthalmology, Inc.