The aims of this study were to (1) determine the reproducibility of sub-maximal and maximal versions of the Yo-Yo intermittent endurance test level 2 (Yo-Yo IE2 test), (2) assess the relationship between the Yo-Yo IE2 test and match performance and (3) quantify the sensitivity of the Yo-Yo IE2 test to detect test-retest changes and discriminate between performance for different playing standards and positions in elite soccer. Elite (n = 148) and sub-elite male (n = 14) soccer players carried out the Yo-Yo IE2 test on several occasions over consecutive seasons. Test-retest coefficient of variation (CV) in Yo-Yo IE2 test performance and heart rate after 6 min were 3.9% (n = 37) and 1.4% (n = 32), respectively. Elite male senior and youth U19 players Yo-Yo IE2 performances were better (P < 0.01) than elite youth U16s and sub-elite players (2,603 ± 451 and 2,534 ± 549 vs. 1,855 ± 535 vs. 1,749 ± 382 m). The intra- and inter-season CV for Yo-Yo IE2 test performance were 4.2 and 5.6%, respectively. A correlation was observed (P < 0.05) between Yo-Yo IE2 test performance and the total (r = 0.74) and high-intensity (r = 0.58) running distance covered in a match. A correlation was also evident (P < 0.01) between Yo-Yo IE2 test heart rate after 6 min expressed in percentage of maximal heart rate and the peak values for high-intensity running performed by midfielders in 5-min (r = -0.71), 15-min (r = -0.75) and 45-min periods (r = -0.77). The present data demonstrate that the Yo-Yo IE2 test is reproducible and can be used to determine the capacity of elite soccer players to perform intense intermittent exercise. Furthermore, the Yo-Yo IE2 test was shown to be a sensitive tool that not only relates to match performance but can also differentiate between intermittent exercise performance of players in various standards, stages of the season and playing positions.