The study aimed to determine the optimal application of single- and paired-pulse transcranial magnetic stimulation (TMS) in the rectus femoris. Twenty-nine male adults participated in the study, which involved 5 separate experiments. Experiments 1 to 3 assessed the effect of conditioning stimulus (CS) intensity (60, 70, 80 and 90% active motor threshold, AMT), contraction strength (5, 10, 20 and 50% maximum voluntary contraction, MVC), and inter-stimulus interval (ISI, 2–5 ms for short-interval intracortical inhibition, SICI and 10–15 ms for intracortical facilitation, ICF) on SICI and ICF. In Experiment 4, 30 measurements of corticospinal excitability (CSE), SICI and ICF were recorded, with the minimum number of consecutive measurements required as a probability of falling within the 95% CI determined. In Experiment 5, within- and between-day reliability of CSE, SICI and ICF was assessed. The results suggest that for SICI, a CS of 70% AMT, ISI of 2 ms, and contraction strength of 5 or 10% MVC induces the greatest level of inhibition. Negligible differences in ICF were seen across stimulus variables. The minimum number of measurements required to obtain an accurate estimate of CSE, SICI and ICF was 21, 18 and 17, respectively. Using the optimal stimulus variables and number of measurements, CSE, SICI and ICF can be measured reliably both within- and between-days (intraclass correlation coefficient, ICC ≥ 0.87, ≥0.74, and ≥0.61, respectively). The current findings can be used to guide future investigations using single- and paired-pulse TMS to elicit responses in the rectus femoris.