This study aims to characterize motor unit (MU) features associated with muscle fatigue, using high-density surface electromyography (HD-sEMG). The same MUs recruited before/after, and during muscle fatigue were identified for analysis. The surface location of the innervation zones (IZs) of the MUs was identified from the HD-sEMG bipolar motor unit action potential (MUAP) map. The depth of the MU was also identified from the decay pattern of the MUAP along the muscle fiber transverse direction. Both the surface IZ location and the MU depth information were utilized to ensure the same MU was examined during the contraction before/after muscle fatigue. The MUAP similarity, defined as the correlation coefficient between MUAP morphology, was adopted to reveal the alterations in MU characteristics under the condition of fatigue. The biomarkers of the same MUs were compared before/after fatigue (task 1) at 5%, 10%, and 15% maximal voluntary contraction (MVC) and in the process of continuous fatigue (task 2) at 20% MVC. Our results indicate that the MUAP morphology similarity of the same MUs was 0.91 ± 0.06 (task 1) and 0.93 ± 0.04 (task 2). The results showed that MUAP morphology maintained good stability before/after, and during muscle fatigue. The findings of this study may advance our understanding of the mechanism of MU neuromuscular fatigue.