Purpose <p>The purpose of this study was to evaluate the effect of repeat testing on N1 and P1 amplitudes, signal-to-noise (SNR) ratios, and amplitude ring ratios (RR) in multifocal electroretinography (mfERG).</p> Methods <p>This was a retrospective review of mfERG records from 08/2022 to 05/2023. Patients were tested binocularly with the Espion system (Diagnosys LLC). Only records from patients with repeat mfERG tests at the same appointment were included. N1 and P1 amplitudes, signal-to-noise ratio (SNR), and amplitude ring ratios were evaluated for the first recording (run #1), the second recording (run #2), and the combination of run #1 and run #2 (combined run).</p> Results <p>Data was collected for 93 eyes from 47 patients (5 males, 42 females) with a mean patient age of 56.1 ± 17.3 years. No change was observed between run #1 and run #2 for N1 or P1 amplitudes, however amplitudes of the combined run decreased significantly (p&#xa0; &lt; &#xa0;0.05) compared to run #1 amplitudes for all rings, right and left eyes (except for ring 1 in right eyes). SNR increased significantly from run #1 to run #2 for rings 2–5 (~10%), but not for ring 1. The number of blinks recorded during testing decreased from run #1 to run #2 (p &lt; 0.001). Amplitude ring ratios R5/R4 and R5/R3 did not change significantly from run #1 to run #2, while amplitude R1/R2 ratio decreased significantly (p &lt; 0.05).</p> Conclusion <p>The change in signal quality from run #1 to run #2 suggests a superior quality signal in the second run. Furthermore, the decreased P1 amplitude in the combined run compared to run #1 should be considered when a clinician uses the combined run for their final report.</p>