We examined the effect of prolonged tennis playing on lower limb muscles' activity during the execution of first and second tennis serves. Ten male competitive tennis players executed five first and second serves before (pretest) and after (posttest) a 3-h tennis match. Surface electromyographic (EMG) activity of four lower limb muscles (vastus lateralis, rectus femoris, gastrocnemius lateralis, and soleus muscles) on each leg was recorded along with maximum ball velocity measured by a radar gun and peak vertical forces recorded by a force platform. For the vastus lateralis, gastrocnemius lateralis, and soleus muscles of the left leg as well as the vastus lateralis muscle of the right leg, EMG amplitude decreased from pre- to posttests (p ≤ 0.033). These reductions in the EMG signal were generally more pronounced in the first serve (i.e., ranging from -10% to -40%) compared to the second serve (0% to -25%). Maximum ball velocity for both first (159 ± 12 vs. 154 ± 12 km/h) and second (126 ± 20 vs. 125 ± 15 km/h) serves remained unchanged from pre- to posttests (p = 0.638) Similarly, peak vertical forces did not differ between pretest and posttest for both first (1.78 ± 0.30 vs. 1.72 ± 0.29 body weight) and second (1.62 ± 0.25 vs. 1.75 ± 0.23 body weight) serves (p = 0.730). In conclusion, a 3-h tennis match led to decreased activation levels in various leg muscles during serves, particularly in first serves compared to second serves. Despite consistent maximum ball velocity and peak vertical forces, these reductions in EMG signals suggest that skilled tennis players may adopt compensatory strategies after prolonged play.