The aim of this work is to assess an automatic optimized algorithm for the positioning of the Motor Units (MUs) within a multilayered cylindrical High Density surface EMG (HD-sEMG) generation model representing a skeletal muscle. The multilayered cylinder is composed of three layers: muscle, adipose and skin tissues. For this purpose, two different algorithms will be compared: an unconstrained random and a Mitchell's Best Candidate (MBC) placements, both with uniform distribution for the MUs positions. These algorithms will then be compared by their fiber density within the muscle and by using a classical amplitude descriptor, the Root-Mean-Square (RMS) amplitude value obtained from 64 HD-sEMG signals recorded by an 8×8 electrode grid of circular electrode during one contraction at 70% Maximum Voluntary Contraction (MVC) in both simulation and experimental conditions for the Biceps Brachii (BB) muscle. The obtained results clearly exposed the necessity to use a specific algorithm to place the MUs within the muscle representation volume in agreement with physiology.