We investigated the effects of familiarization on the reliability of gross efficiency (GE) and lower-limb electromyography (EMG-rms) of adults and adolescents. We also evaluated the relationship between inter-test differences in GE and EMG-rms. Nine adult and nine adolescent cyclists performed three 10-min cycling tests at 50% of peak power output, separated by 48-h. 45-min familiarization visits were performed 24-h after each test. No differences were found across the tests for adult cyclists' GE or EMG-rms, with Coefficient of Variation (CV%) ranging from 2.6% - 2.9% (GE) and 4.3% - 7.4%. Among adolescents, there was an increase in GE between tests 1 and 2 (P 0.05) between the inter-test differences of GE and EMG-rms. GE can be reliably determined; however, adolescents require two exposures to cycling. Without familiarization, adolescent EMG-rms is more variable than adults and would require larger samples to establish differences. The weak relationships between inter-test EMG-rms and inter-test GE questions the link between muscle activation and GE changes.