Purpose: The effect of playing surface on physical performance during a repeated-sprint ability (RSA) test and the mechanisms for any potential playing-surface-dependent effects on RSA performance are equivocal. The purpose of this study was to investigate the effect of natural grass (NG) and artificial turf (AT) on physical performance, ratings of perceived exertion, feeling scale, and blood biomarkers related to anaerobic contribution (blood lactate [Lac]), muscle damage (creatine kinase and lactate dehydrogenase), inflammation (C-reactive protein), and immune function (neutrophils [NEU], lymphocytes [LYM], and monocytes) in response to an RSA test. Methods: A total of 9 male professional football players from the same regional team completed 2 sessions of RSA testing (6 × 30 s interspersed with a 35-s recovery) on NG and AT in a randomized order. During the RSA test, total (sum of distances) and peak (highest distance covered in a single repetition) distance covered were determined using a measuring tape, and the decrement in sprinting performance from the first to the last repetition was calculated. Before and after the RSA test, ratings of perceived exertion, feeling scale, and Lac, creatine kinase, lactate dehydrogenase, C-reactive protein, NEU, LYM, and monocytes were recorded in both NG and AT conditions. Results: Although physical performance declined during the RSA blocks on both surfaces (P = .001), the distance covered declined more on NG (15%) than on AT (11%; P = .04; effect size [ES] = −0.34; 95% confidence interval [CI], −1.21 to 0.56) with a higher total distance covered (+6% [2%]) on AT (P = .018; ES = 1.15; 95% CI, 0.16 to 2.04). In addition, lower ratings of perceived exertion (P = .04; ES = −0.49; 95% CI, −1.36 to 0.42), Lac, NEU, and LYM (P = .03; ES = −0.80; 95% CI, −1.67 to 0.14; ES = −0.16; 95% CI, −1.03 to 0.72; and ES = −0.94; 95% CI, −1.82 to 0.02, respectively) and more positive feelings (P = .02; ES = 0.81; 95% CI, −0.13 to 1.69) were observed after the RSA test performed on AT than on NG. No differences were observed in the remaining physical and blood markers. Conclusion: These findings suggest that RSA performance is enhanced on AT compared with NG. This effect was accompanied by lower fatigue perception and Lac, NEU, and LYM and a more pleasurable feeling. These observations might have implications for physical performance in intermittent team-sport athletes who train and compete on different playing surfaces.