Purpose:The purpose of this study was to investigate the effect of interstimulus frequency on the photopic negative response (PhNR) in the clinical electroretinogram (ERG) in glaucoma and healthy eyes. Methods:Participants with open angle glaucoma (n = 15) and age-matched controls (n = 20) were recruited. Photopic ERGs were recorded in one eye using five frequencies (1-5 Hz) delivered in random order. ERGs were analyzed for changes to amplitude and timing between groups and interstimulus frequency. Coefficient of variation (CoV) was used to examine variability within recordings for each frequency. Results:While the a-wave and b-wave showed minimal alteration, the PhNR was highly sensitive to changes in interstimulus frequency. The PhNR signal was largest at 1 Hz and steadily diminished with higher frequencies in both control and glaucoma groups. Significant differences in PhNR amplitude were found between controls and glaucoma groups at 2 and 3 Hz. While 1 Hz delivered the largest PhNR, it also showed a significantly greater CoV compared to other frequencies. Conclusions:An interstimulus frequency of 2 Hz was optimal for recording the PhNR, creating a good balance between testing time and signal quality. A higher frequency could be used to further shorten clinical testing times; however, this may compromise its clinical utility by dampening the PhNR. Translational Relevance:Here we show the importance of considering flash interstimulus frequency when designing ERG protocols for recording the PhNR as while higher frequencies can shorten test times, they also have considerable effects on the PhNR.