Adaptive optics scanning light ophthalmoscopy (AOSLO) enables high-resolution retinal imaging, eye tracking, and stimulus delivery in the living eye. AOSLO-mediated visual stimuli are created by temporally modulating the excitation light as it scans across the retina. As a result, each location within the field of view receives a brief flash of light during each scanner cycle (every 33-40 ms). Here we used in vivo calcium imaging with AOSLO to investigate the impact of this intermittent stimulation on the retinal ON and OFF pathways. Raster-scanned backgrounds exaggerated existing ON-OFF pathway asymmetries leading to high baseline activity in ON cells and increased response rectification in OFF cells.