The aims of this study were to evaluate physical performance of substitute players versus those replaced or completing the entire match, determine physical performance of substitute players across different playing positions and examine variations in match-related running performance in substitute players throughout the entire competitive season. The sample was composed of 943 observations of professional players who participated in the first division of the Spanish League (La Liga) during the 2014-2015 season. The players were divided into three different groups: players who completed the entire match (n = 519), players who were replaced (n = 212) and substitute players (n = 212). Substitute players covered greater distances at medium and high intensity compared to the players who played the entire match and those who were replaced. Position-specific trends indicated that attackers and central midfielder increased the distance covered at high-intensity running compared to their peers who played the whole match. During the competitive season, it was observed that substitute players attained greater match running performance during the mid-season period, allowing them to cover more distance for different variables of running performance compared to the start and end of the season.