Although the behaviour of individual motor units is classically studied with intramuscular EMG, recently developed techniques allow its analysis also from EMG recorded in multiple locations over the skin surface (high-density surface EMG). The analysis of motor units from the surface EMG is useful when the insertion of needles is not desirable or not possible. Moreover, surface EMG allows the measure of motor unit properties which are difficult to assess with invasive technology (e.g., muscle fiber conduction velocity or location of innervation zones) and may increase the number of detectable motor units with respect to selective intramuscular recordings. Although some limitations remain, both the discharge pattern and muscle fiber properties of individual motor units can currently be analyzed non-invasively. This review presents the conditions and methodologies which allow the investigation of motor units with surface EMG.