We assessed lower limb muscle activity during the execution of first and second tennis serves, exploring whether the extent of these differences is influenced by the chosen method for normalising surface electromyography (EMG) data. Ten male competitive tennis players first completed three rounds of maximal isometric voluntary contractions (MVC) of knee extensors and plantar flexors for the left (front) and right (back) leg separately, and three squat jumps. Afterward, they executed ten first and ten-second serves. Surface EMG activity of four lower limb muscles (vastus lateralis, rectus femoris, gastrocnemius lateralis, and soleus muscles) on each leg was recorded and normalised in three different ways: to MVC; to peak/maximal activity measured during squat jump; and to the actual serve. For the rectus femoris and soleus muscles of the left leg, and the gastrocnemius lateralis and soleus muscles of the right leg, EMG amplitude differed significantly between normalisation techniques (P ≤ 0.012). All muscles showed greater activity during the first serve, although this difference was only statistically significant for the right vastus lateralis muscle (P = 0.014). In conclusion, the EMG normalisation method selected may offer similar information when comparing first and second serve, at least for leg muscles studied here.