This study investigated the incidence of injury in sub-elite Rugby League players before and after the introduction of the limited interchange rule. The incidence of injury was prospectively studied in one sub-elite Rugby League club over three competitive seasons. Two seasons were played under the unlimited interchange rule, while the third season was played under the limited interchange rule. Players participated in 91 matches under the unlimited interchange rule and 44 matches under the limited interchange rule. Injury was defined as any pain, disability or injury that occurred as a result of a competition game that caused the player to miss a subsequent game. The relative risk (RR) of injury significantly decreased (RR = 0.70 [0.65 to 0.75], P<0.05) following the introduction of the limited interchange rule, with the incidence of injury decreasing from 72.5 (58.2 to 86.8) per 1000 playing hr under the unlimited interchange rule to 51.0 (33.8 to 68.1) per 1000 playing hr under the limited interchange rule. The risk of sustaining thigh and calf injuries (RR= 0.27 [0.12 to 0.59], P < 0.05), muscular strains (RR= 0.23 10.17 to 0.31]., P<0.05), and high intensity running injuries (RR= 0.16 [0.04 to 0.691, P<0.05) was significantly reduced following the introduction of the limited interchange rule. These findings demonstrate that the risk of injury in sub-elite Rugby League players is significantly reduced following the introduction of the limited interchange rule. The reduced injury risk may reflect a fatigue-induced reduction in match speed and impact forces associated with physical collisions and tackles.