The purpose of this study was to evaluate the reliability and reproducibility of the physiological and overload features of the Yo-Yo intermittent endurance test level 2 (Yo-Yo IE2) in competitive male soccer (n = 20), basketball (n = 11), and volleyball players (n = 10). The participants completed Yo-Yo IE2 tests on three separate occasions with assessment of performance, heart rate, running speed, accelerations, decelerations and body load using GPS instrumentation. The intra-class correlation coefficient index, confidence intervals and coefficients of variation were calculated to assess the reliability of the test. Intra-class correlation coefficients for test-retest trials in the total sample ranged from large to nearly perfect (total distance: 0.896; mean speed: 0.535; maximum speed: 0.715; mean HR: 0.876; maximum HR: 0.866; body load: 0.865). The coefficients of variation for distance, mean speed, HR response, as well as acceleration and deceleration scores for test-retest trials ranged from 1.2 to 12.5% with no differences observed among particular sport disciplines. The CV for shuttles performed ranged from 4.4 to 5.5% in all sports. Similar results were obtained for the three different categories of players tested. These results suggest that the Yo-Yo IE2 test appears to be a reliable alternative for evaluating the ability to perform intermittent high-intensity running in different outdoor and indoor team sports. Players may need one or two familiarization tests to ensure valid assessment of intermittent endurance capacity. It appears that the Yo-Yo IE2 test incorporates accelerations and decelerations in a consistent and reproducible fashion.