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