Decomposition of the surface electromyographic (sEMG) signal is commonly used to examine motor unit (MU) firing behavior. However, the intra- and inter-day reliability of these measurements has yet to be quantified or reported. This investigation 1) examined the effect of input excitation on the mean firing rate (MFR) vs. recruitment threshold (RT) relationship and 2) determined the inter- and intra-day reliability of the MFR vs. RT relationship at 30%, 50%, and 70% of maximum voluntary isometric strength (MVIC). Twenty-eight healthy males (23 ± 3 yr) completed two experimental visits, during which they performed MVIC testing and isometric ramp contractions at 30%, 50%, and 70% MVIC. sEMG signals were recorded from the vastus lateralis during the ramp contractions and decomposed to establish the MFR vs. RT relationship for the detected MUs. Intra- and inter-day reliability was then established for the slopes and y-intercepts of the MFR vs. RT relationship at each contraction intensity. All participants displayed significant MFR vs. RT relationships ( r range: −0.662 to −0.999; P ≤ 0.001–0.006). Intra- and inter-day intraclass correlation coefficients (ICCs) ranged from 0.766–0.824 and 0.867–0.919 for the slopes and from 0.780–0.915 and 0.804–0.927 for the y-intercepts, respectively. Furthermore, the slope coefficient was significantly greater at 70% than at 30% MVIC, and the y-intercepts increased with increasing contraction intensities. Changes in input excitation to the MU pool alter the magnitude, but not the reliability, of the slopes and y-intercepts of the MFR vs. RT relationship. NEW & NOTEWORTHY The firing behavior of the motor unit (MU) pool is often characterized using the mean firing rate vs. recruitment threshold relationship of the active MUs. Although this relationship has been widely used, this is the first study to report the effects of input excitation (contraction intensity) on the intra- and inter-day reliability of this relationship. The criteria used for MU analysis and the model utilized in this study allow for generalization to outside investigators and laboratories.