Characterization of fatigue using surface electromyography (sEMG) data has been motivated for rehabilitation and injury-preventative technologies. Current sEMG-based models of fatigue are limited due to (a) linear and parametric assumptions, (b) lack of a holistic neurophysiological view, and (c) complex and heterogeneous responses. This paper proposes and validates a data-driven non-parametric functional muscle network analysis to reliably characterize fatigue-related changes in synergistic muscle coordination and distribution of neural drive at the peripheral level. The proposed approach was tested on data collected in this study from the lower extremities of 26 asymptomatic volunteers (13 subjects were assigned to the fatigue intervention group, and 13 age/gender-matched subjects were assigned to the control group). Volitional fatigue was induced in the intervention group by moderate-intensity unilateral leg press exercises. The proposed non-parametric functional muscle network demonstrated a consistent decrease in connectivity after the fatigue intervention, as indicated by network degree, weighted clustering coefficient (WCC), and global efficiency. The graph metrics displayed consistent and significant decreases at the group level, individual subject level, and individual muscle level. For the first time, this paper proposed a non-parametric functional muscle network and highlighted the corresponding potential as a sensitive biomarker of fatigue with superior performance to conventional spectrotemporal measures.