This study aimed to analyse the effect of playing surface temperature on muscular and thermal response to a repeated-sprint ability (RSA) test in football players. Thirty-two male football players (23 ± 5 years; 1.77 ± 0.06 m; 71.2 ± 6.7 kg) from two squads of a third-division football club participated in the study. An RSA test was carried out at a high surface temperature (45.34 ± 2.53 °C) and low surface temperature (27.21 ± 2.17 °C). Before and after this test, the muscular response of the players was assessed through tensiomyography and thermograms. The results revealed that performance in the RSA test particularly increased at a higher surface temperature, especially in the first 5 m of the 30 m sprint test. While a reduction in maximal radial displacement (Dm) in the biceps femoris post-RSA was observed at lower surface temperatures, a higher temperature on the thigh, hamstring and calf was found in the higher surface temperature group. In conclusion, higher surface temperatures had an influence on players' thermal and tensiomyographic profile and improved performance in their repeated-sprint ability. These results suggest a need for coaches and players to be aware of these parameters to ensure adequate functionality and safety of the playing surface.