Abstract Aims Impaired vision is an additional risk factor in elderly for falls. We investigated the hypothesis that treadmill (TM) walking affects visual function in both healthy elderly and those with early-moderate visual dysfunction due to glaucoma. Methods Thirty healthy controls (HC) aged 64–83 years and 18 glaucoma patients (GLA) aged 62–82 years participated in this cross-sectional study. The impact of TM-walking on visual function was assessed binocularly for (i) best-corrected visual acuity (BCVA) with and without crowding effect, (ii) contrast sensitivity (CS), and (iii) and visual field (mean deviation, VF-MD). Visual function was tested while participants were standing or during TM-walking for 2 speed conditions: (i) fast walking at their preferred speed and (ii) walking at a fixed speed of 3.5 km/h. Results GLA, most with early-moderate VF loss, performed equally well as HC. Independent of GROUP, an impact of SPEED on visual functions was statistically evident with large statistical effect size for (i) both types of BCVA with a mean loss of 0.02–0.05 logMAR (η2 = 0.41) and (ii) VF-MD with mean loss of 1 dB (η2 = 0.70), but not for CS. Conclusions Here, we introduce a paradigm for the assessment of visual function during walking. We provide proof-of-concept that our approach allows for the identification of walking induced visual function loss, i.e., a deterioration of BCVA and VF-sensitivity during TM-walking in both groups. It is therefore of promise for the investigation of the relation of vision impairment and mobility, ultimately the increased frequency of falls in advanced glaucoma.