This study examined motor unit (MU) amplitudes (AP) and firing rates during moderate-intensity contractions and muscle cross-sectional area (mCSA) and echo intensity (mEI) of the vastus lateralis (VL) in chronically endurance-trained and sedentary females. Eight endurance-trained (ET) and nine sedentary controls (SED) volunteered for this study. Surface electromyographic (EMG) signals from a five-pin electrode array were recorded from the VL during isometric trapezoid muscle actions at 40% of maximal voluntary contraction (MVC). Decomposition methods were applied to the EMG signals to extract the firing events and amplitudes of single MUs. The mean firing rate (MFR) during steady force and MUAP for each MU was regressed against recruitment threshold (RT, expressed as %MVC). The y-intercepts and slopes from the MFR and MUAP vs. RT relationships were calculated. EMG amplitude during steady force was normalized (N-EMG) to peak EMG amplitude recorded during the MVC. Ultrasonography was used to measure mCSA and mEI. Significant differences existed between the ET and SED for the slopes (P = 0.005, P = 0.001) from the MFR and MUAP vs. RT relationships with no differences for the y-intercepts (P > 0.05). N-EMG was significantly (P = 0.033) lower for the ET than SED. There were no differences between groups for mCSA; however, the SED possessed significantly (P = 0.001) greater mEI. Subsequently, the ET likely possessed hypertrophied and stronger MUs that allowed for lower necessary muscle activation to maintain the same relative task as the SED. The larger MUs for the ET is supported via the MFR vs. RT relationships and ultrasound data.