The aim of this study was to investigate the effect of leg preference and cycling experience on unilateral muscle efficiency and muscle activation. To achieve this purpose, two experiments were performed. Experiment 1 involved eight cyclists and experiment 2 included eight non-cyclists. Subjects underwent an incremental maximal test and submaximal trials of one-legged cycling for preferred and non-preferred leg. Oxygen uptake and muscle efficiency were compared between legs. The magnitude of muscle activation (RMS) and the inter-limb excitation were monitored for the vastus lateralis, biceps femoris and gastrocnemius (medial head) muscles during one-legged cycling with preferred and non-preferred leg. Variables of muscle activation, oxygen uptake and muscle efficiency (gross and net) did not differ between legs (P>0.05). The magnitude of muscle activation and its variability were similar between legs while performing the unilateral pedaling. Inter-limb communication did not differ between experiments (P>0.05). Similar activation between legs was consistent with the influence of bilateral practice for attaining similar performance between sides. These results do not support asymmetry in magnitude of muscle activation during pedaling.