The purpose of this study was to quantify the effect of different starting distances on 5-m sprint time and the accuracy of the initial timing gate. A single-beam timing gate system (1 m high) was used to measure 5-m sprint time in 13 male sports students. Each subject performed 3 valid trials for 3 starting distances: 0.3, 0.5, and 1.0 m from the initial timing lights, respectively. A high-speed video camera was used to track a reflective marker placed on the subjects' hip within a field of view around the initial timing gate. Accuracy of the initial timing gate was defined as the time between the initial timing light trigger and passing of the reflective marker by the initial timing gate. Sprint times were significantly faster for the 1.0-m starting distance (0.98 ± 0.06 seconds) than for the 0.5-m (1.05 ± 0.07 seconds) and the 0.3-m (1.09 ± 0.08 seconds) starting distances (p < 0.001). There were no differences in initial timing gate error between starting distances (p = 0.078). Hence, starting distance influenced sprint times but not the accuracy of the initial timing gate. Researchers and coaches should consider the effect of starting distance on 5-m sprint time and ensure consistent testing protocols. Based on the results of this study, we recommend a starting distance of 0.3 m that should be used for all sprint performance tests.