The aim of this study was to determine the effect of prior cycling on EMG activity of selected lower leg muscles during running. Ten elite level triathletes underwent two testing sessions at race pace: a 40 km cycle followed by a 2 km run (CR) and a 10 km run followed by a 2 km run (RR). EMG data from selected lower limb muscles were collected at three sections of each run (0 km, 1 km and 2 km) for six strides using a portable data logger. Significant differences (p < 0.05) between condition were found for the level of activation (Lact) for biceps femoris (BF) during stance and vastus lateralis (VL) during flight and stance. Vastus medialis (VM) changed in Lact, during flight, between sections in the 2 km run. Furthermore, significant differences (p < 0.05) between condition were found for BF during stance and for rectus femoris (RF) and VM during flight. There was a significant difference (p < 0.05) in the duration of VL activation (Dact) across sections of the 2 km run. Findings from this investigation highlight changes in muscle function when changing from cycling to running and indicate a need to train specifically for the cycle to run transition. Such training may improve performance and reduce the risk of injury.