PURPOSE: The application of horizontal resistance is key in sprint training to modulate velocity and promote adaptations. However, the interaction between velocity and resistance on muscle excitation remains insufficiently studied. This study assessed the electromyographic (EMG) activity of thigh and gluteal muscles in response to varying velocity and resistance during sprinting. METHODS: Thirty-seven elite athletes (27 females and 10 males) performed two trials under three conditions: a 40-meter maximal sprint without resistance and two resisted sprints applied by a robotic device with resistive forces equivalent to 25% and 75% of body mass in a sled condition. EMG activity was recorded from eight muscles in the quadriceps, hamstrings, and gluteal muscle groups for both lower limbs. Generalized mixed models were used to analyze average EMG changes with increasing velocity as a function of resistance. Statistical parametric mapping was used to assess the changes within the stance and swing phases as a function of sprint phase and resistance. RESULTS: Hamstring EMG activity increased with increasing velocity (+17.9% in stance phase without resistance) and decreased as resistance increased, with opposite effects observed in quadriceps muscles. Higher gluteal EMG activity was found during the late swing phase with increasing velocity and decreasing resistance. The biceps femoris long head exhibited the largest EMG increase with velocity (+23% in the stance phase) among hamstrings, while the semitendinosus showed the highest increase with reduced resistance (+27% in the stance phase). CONCLUSIONS: External resistance and running speed influence thigh muscle activity differently, even within the same muscle group, likely due to distinct muscle architecture and function. These insights should be considered when designing sprint training programs to target specific muscle groups, avoid muscle overloading, and account for inter-individual differences to optimize performance and reduce injury risk.