Despite its increasing use in research, High-Density EMG (HD-EMG) has yet to gain widespread acceptance in routine clinical evaluations, particularly for assessing age-related variations and central nervous system disorder. This pilot study aims to evaluate the feasibility of using a portable HD-EMG system during daily rehabilitation tests with the Lokomat, a robotic platform widely used in neurorehabilitation, to study MU properties in different populations. Nine participants, categorized in three groups (healthy young, healthy elderly and individual with chronic stroke) were recruited. Participants performed isometric sub-maximal contractions at three force levels within the Lokomat, and myoelectrical signals were acquired through a portable 64-channel HD-EMG system. Our findings suggest that traditional sEMG amplitude and spectral parameters fail to identify differences among healthy control groups, whereas HD-EMG can effectively capture age-related variations through the analysis of MU properties (i.e., number of extracted MUs). Additionally, in contrast to the traditional parameters typically used by clinicians, MU property analysis revealed distinct neuromuscular alterations when comparing individuals who had a stroke with healthy groups. Results highlight the potential of HD-EMG in exploring specific neuromuscular changes associated with stroke. Therefore, this pilot study shows the feasibility of integrating HD-EMG into clinical test sessions with the Lokomat. By studying MU properties, HD-EMG provides valuable insights into muscle behavior, potentially advancing our understanding of neuromuscular conditions and effectively optimizing rehabilitation treatments. We believe that further research in this direction is needed to establish HD-EMG as a standard clinical diagnostic tool.