Optomotor response is increasingly used in preclinical research for evaluating the visual function in rodents. However, the most suitable measuring protocol for specific scientific questions is not always established. We aimed to determine the optimal parameters for visual function analysis in experimental autoimmune encephalomyelitis optic neuritis (EAEON), an animal model for multiple sclerosis. Contrast sensitivity as well as spatial frequency both had a low variance and a good test-retest reliability. Also, both parameters were able to differentiate between the EAEON and the control group. Correlations with the retinal degeneration, assessed by optical coherence tomography, the infiltration of immune cells, and the clinical disability score revealed that spatial frequency was superior to contrast sensitivity analysis. We therefore conclude that spatial frequency testing is better suited as visual acuity assessment in C57Bl/6 J EAEON mice. Furthermore, contrast sensitivity measurements are more time consuming, possibly leading to more stress for the animals.