Motor unit action potential (MUP) analysis in human medicine is a valuable and important diagnostic technique enabling discrimination between myogenic and neurogenic problems. This study establishes normative data in subclavian, triceps and lateral vastus muscles for clinical application of MUP analysis in the Warmblood horse, and examines whether muscle differences are present. Electromyographic (EMG) needle examination and MUP analysis were performed of the triceps, lateral vastus and subclavian muscles in 7 awake, nonsedated, Warmblood horses age 4–10 years. The amplitude, duration, number of phases and turns were calculated from the recorded superimposed MUPs together with intramuscular and rectal temperatures. No significant differences were found in duration of insertional activity between the 3 muscles. The mean ± s.d. duration of the insertional activity was 526 ± 148.3 ms. The MUP amplitude of all 3 muscles differed significantly, with the highest amplitude (427 ± 3.20 μV) in the triceps and the lowest (220 ± 2.08 μV) in the subclavian muscle. The number of turns of the lateral vastus (3.0 ± 1.22) was significantly higher than that of the triceps muscle (2.7 ± 1.51). No differences were found in MUP duration (5.9–6.4 ms).