Purpose: The purposes of the present study were: (1) to determine whether the physical working capacity at the fatigue threshold (PWCFT) model that has been used for estimating the onset of neuromuscular fatigue in the vastus lateralis (VL) during incremental treadmill running could also be applied to the vastus medialis (VM), biceps femoris (BF), and semitendinosus (ST) muscles; and (2) if applicable, to compare the running velocities associated with the PWCFT among these muscles. Methods: Eleven subjects (age 21.7 +/- 1.8 years) performed an incremental treadmill test to exhaustion with electromyographic signals recorded from the VL, VM, BF, and ST. Results: The results indicated there were no significant (p > 0.05) mean differences in the running velocities associated with the PWCFT for the VL (14.4 +/- 2.0 km/h), VM (14.3 +/- 1.9 km/h), BF (13.8 +/- 1.8 km/h), and ST (14.7 +/- 2.3 km/h). In addition, there were significant inter-correlations (r = 0.68-0.88) among running velocities associated with the PWCFT of each muscle. Individual results also indicated that 9 of the 11 subjects exhibited identical PWCFT values for at least 3 of the 4 muscles, but there were no uniform patterns for any intra-individual differences. Conclusion: The findings of the present study suggested that the PWCFT test is a viable method to identify neuromuscular fatigue in the quadriceps and hamstrings during incremental treadmill exercise and results in consistent PWCFT values among these muscles.