A frequently observed local measurement artifact with spectral domain OCT is caused by the void signal of the retinal vasculature. This study investigated the effect of suppression of blood vessel artifacts with and without retinal hyperaemia. Spectral domain OCT scans, centred on the optic nerve head, were performed in 46 healthy subjects (92 eyes). Baseline scans were made during rest, while for the follow-up scan, 23 subjects (50 %) performed strenuous physical exercise. Systemic and retinal hyperaemia were quantified. Quantification of retinal nerve fibre layer (RNFL) thickness was performed with and without suppression of retinal blood vessel artifacts. The potential systematic effect on RNFL thickness measurements was analysed using Bland-Altman plots. At baseline (no retinal hyperaemia), there was a systematic difference in RNFL thickness (3.4 μm, limits of agreement -0.9 to 7.7) with higher values if blood vessel artifacts were not suppressed. There was significant retinal hyperaemia in the exercise group (p < 0.0001). Baseline thickness increased from 93.18 to 93.83 μm (p < 0.05) in the exercise group using the algorithm with blood vessel artifact suppression, but no significant changes were observed using the algorithm without blood vessel artifact suppression. Retinal hyperaemia leads to blood vessel artifacts which are relevant to the precision of OCT layer segmentation algorithms. The two algorithms investigated in this study can not be used interchangeably. The algorithm with blood vessel artifact suppression was more sensitive in detecting small changes in RNFL thickness. This may be relevant for the use of OCT in a range of neurodegenerative diseases were only a small degree of retinal layer atrophy have been found so far.