The purpose of the present study was to determine the reliability of a 5km run test on a motorized treadmill. Over three consecutive weeks, 12 well-trained runners completed three 5km time trials on a treadmill following a standardized warm-up. Runners were partially-blinded to their running speed and distance covered. Total time to complete the run was recorded for analysis of reliability. The highest intraclass correlation coefficient between trials for 5km time trial (TT) time was .99 (90% confidence intervals [CI]; .96–1.00, very high), which occurred between trials 2 and 3. The lowest typical error of measurement (expressed as a coefficient of variation percentage [CV%] and as an absolute value in seconds) also occurred between trials 2 and 3, and was 1.0% and 10.9 seconds, respectively. The testing protocol performed on a motorized treadmill in the current study is reproducible in well-trained runners following a single trial, making it a reliable method for monitoring running performance.