The COVID-19 pandemic interrupted professional football in the 2019/2020 season, and football experts anticipate that the consequences of lockdown measures will negatively affect the physical performance of players once competition restarts. This study aimed to evaluate position-specific match running performance (MRP) to determine the effect of COVID-19 lockdowns on the physical performance of professional football players. Players’ MRPs (n = 124) were observed in matches before and after the COVID-19 lockdown in the 2019/2020 season of the highest level of national competition in Croatia and were classified according to player position: central defenders (CD; n = 42), fullbacks (FB; n = 20), midfielders (MF; n = 46), and forwards (FW; n = 16). The MRPs were measured using Global Positioning System, and included the total distance covered, low-intensity running (≤14.3 km/h), running (14.4–19.7 km/h), high-intensity running (≥19.8 km/h), total accelerations (>0.5 m/s2), high-intensity accelerations (>3 m/s2), total decelerations (less than –0.5 m/s2), and high-intensity decelerations (less than –3 m/s2). The results indicated that, in matches after the COVID-19 lockdown, (i) CDs and FBs featured lower running and high-intensity running (t-value: from 2.05 to 3.51; all p < 0.05; moderate to large effect sizes), (ii) MFs covered a greater distance in low-intensity running and achieved a lower number of total accelerations, and total and high-intensity decelerations (t-value: from –3.54 to 2.46; all p < 0.05, moderate to large effect sizes), and (iii) FWs featured lower high-intensity running (t-value = 2.66, p = 0.02, large effect size). These findings demonstrate that the physical performances of football players from the Croatian first division significantly decreased in matches after the COVID-19 lockdown. A combination of inadequate adaptation to football-specific match demands and a crowded schedule after the competition was restarted most likely resulted in such an effect.