During the early period of running after cycling, EMG patterns of the leg are modified in only some highly trained triathletes. The majority of studies have analysed muscle EMG patterns at arbitrary, predetermined time points. The purpose of this study was to examine changes to EMG patterns of the lower limb at physiologically determined times during the cycle-run transition period to better investigate neuromuscular adaptations. Six highly trained triathletes completed a 10 min isolated run (IR), 30 min of rest, then a 20 min cycling procedure, before a 10 min transition run (C-R). Surface EMG activity of eight lower limb muscles was recorded, normalised and quantified at four time points. Oxygen uptake and heart rate values were also collected. Across all muscles, mean (±SD) EMG patterns, demonstrated significant levels of reproducibility for each participant at all four time points (α < 0.05; r = 0.52–0.97). Mean EMG patterns during C-R correlated highly with the IR patterns (α < 0.05). These results show that EMG patterns during subsequent running are not significantly affected by prior cycling. However, variability of muscle recruitment activity does appear to increase during C-R transition when compared to IR.