The aims of the study were 1) to evaluate whether a multifaceted simulated soccer game protocol, entitled the Copenhagen Soccer Test (CST), elicited a similar physiological loading as a competitive game (CG) and 2) to determine muscle metabolites, blood variables, and sprint performance in various phases of CST. Twelve Danish Second- and Third-Division soccer players participated in the study. On separate days, HR measurements, frequent blood sampling, and physical/technical tests were performed during 60- and 90-min versions of the CST during which repeated musculus vastus lateralis biopsies were collected. A CG was also played, where HR was recorded and pre- and post-game muscle biopsies and blood samples were collected. No differences were observed between CST and CG in average HR (85% ± 1% and 86% ± 1% HRmax, P > 0.05) or recovery plasma creatine kinase (24 h: 312 ± 57 and 324 ± 76 U·L, P > 0.05). Muscle glycogen decreased (P < 0.05) from 459 ± 15 to 232 ± 30 mmol·kg dry weight (d.w.) during CST, which was not different from CG (P > 0.05). The rate of glycogen utilization was 4 ± 1 mmol·kg d.w.·min during the warm-up and the first 15 min of CST and 1 ± 1 mmol·kg d.w.·min (P < 0.05) from 60 to 90 min of CST. After 15 min of CST, muscle lactate was elevated (P < 0.05) approximately fivefold (24 ± 3 mmol·kg d.w.), and creatine phosphate was lowered (P < 0.05) by ∼60% (28 ± 4 mmol·kg d.w.). Sprint velocity (2 × 20 m) decreased (P < 0.05) by 7% during CST (5.2 ± 0.6 to 4.9 ± 0.7 m·s). The physiological response to the CST was reproducible and comparable to that of high-level CG. The CST allowed for rapid muscle sampling and revealed high creatine phosphate degradation throughout the test and a lowered glycogen utilization toward the end of the simulated game.