(1) Background: this study aimed to determine if there are differences in quadriceps and hamstring muscle activation in professional male and female soccer players. (2) Methods: muscle activation was recorded by surface electromyography in 27 professional soccer players (19 male and 8 female). The players performed the Bulgarian squat and lunge exercises. Vastus medialis, vastus lateralis, rectus femoris, semitendinosus, and biceps femoris were the muscles analyzed. (3) Results: The statistical analysis of the hamstring:quadriceps ratio showed no significant differences (p > 0.05). Significant differences were found in the vastus medialis:vastus lateralis ratio for both the lunge exercise (t20 = 3.35; p = 0.001; d = 1.42) and the Bulgarian squat (t23 = 4.15; p < 0.001; d = 1.76). For the intragroup muscular pattern in the lunge and Bulgarian squat exercises, the female players showed higher activation for the vastus lateralis muscle (p < 0.001) than the male players and lower muscle activation in the vastus medialis. No significant differences were found in the rectus femoris, biceps remoris, and semitendinosus muscles (p > 0.05). (4) Conclusions: Differences were found in the medial ratio (vastus medialis: vastus lateralis). Moreover, regarding the intramuscular pattern, very consistent patterns have been found. In the quadriceps muscle: VM>VL>RF; in the hamstring muscle: ST>BF. These patterns could be very useful in the recovery process from an injury to return players to their highest performance.
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