The aim of this study was to interpret changes in experimentally recorded M-waves with progressive motor unit (MU) activation based on simulation of the surface EMG. Activation order during transcutaneous electrical stimulation was analyzed by investigating M-wave average rectified value, spectral properties, and conduction velocity (CV) during electrically elicited contractions. M-waves were detected from the biceps brachii muscle of 10 healthy male subjects by a linear adhesive array of 8 electrodes. Electrical stimulation was delivered to the motor point at either constant current intensity (40, 60, 80, and 100% of the supra-maximal stimulation current) or with linearly increasing current. A model of surface EMG generation that varied activation order based on MU size and location was used to interpret the experimental results. From the experimental and model analysis, it was found that: 1) MUs tended to be activated from low to high CV and from the superficial to the deep muscle layers with increasing transcutaneous electrical stimulation of the biceps brachii muscle; and 2) characteristic spectral frequencies of the M-wave were affected by many factors other than average CV (such as the activation order by MU location or the spread of the MU innervation zones and CVs), thus decreasing with a concomitant increase in CV during progressive MU activation.