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.