Diabetic peripheral neuropathy (DPN) is associated with loss of motor units (MUs), which can cause changes in the activation pattern of muscle fibres. This study investigated the pattern of muscle activation using high-density surface electromyography (HD-sEMG) signals from subjects with type 2 diabetes mellitus (T2DM) and DPN. Thirty-five adults participated in the study: 12 healthy subjects (HV), 12 patients with T2DM without DPN (No-DPN) and 11 patients with T2DM with DPN (DPN). HD-sEMG signals were recorded in the tibialis anterior muscle during an isometric contraction of ankle dorsiflexion at 50% of the maximum voluntary isometric contraction (MVIC) during 30-s. The calculated HD-sEMG signals parameters were the normalised root mean square (RMS), normalised median frequency (MDF), coefficient of variation (CoV) and modified entropy (ME). The RMS increased significantly (p = 0.001) with time only for the DPN group, while the MDF decreased significantly (p < 0.01) with time for the three groups. Moreover, the ME was significantly lower (p = 0.005), and CoV was significantly higher (p = 0.003) for the DPN group than the HV group. Using HD-sEMG, we have demonstrated a reduction in the number of MU recruited by individuals with DPN. This study provides proof of concept for the clinical utility of this technique for identifying neuromuscular impairment caused by DPN.