Abstract Purpose This study examined the repeated bout effect of two resistance training bouts on cycling efficiency and performance. Methods Ten male resistance-untrained cyclists (age 38 ± 13 years; height 180.4 ± 7.0 cm; weight 80.1 ± 10.1; kg; VO2max 51.0 ± 7.6 ml.kg−1.min−1) undertook two resistance training bouts at six-repetition maximum. Blood creatine kinase (CK), delayed-onset of muscle soreness (DOMS), counter-movement jump (CMJ), squat jump (SJ), submaximal cycling and time-trial performance were examined prior to (Tbase), 24 (T24) and 48 (T48) h post each resistance training bout. Results There were significantly lower values for DOMS (p = 0.027) after Bout 2 than Bout 1. No differences were found between bouts for CK, CMJ, SJ and submaximal cycling performance. However, jump height (CMJ and SJ) submaximal cycling measures (ventilation and perceived exertion) were impaired at T24 and T48 compared to Tbase (p < 0.05). Net efficiency during submaximal cycling improved at Bout 2 (23.8 ± 1.2) than Bout 1 (24.3 ± 1.0%). There were no changes in cycling time-trial performance, although segmental differences in cadence were observed between bouts and time (i.e. Tbase vs T24 vs T48; p < 0.05). Conclusion Cyclists improved their cycling efficiency from Bout 1 to Bout 2 possibly due to the repeated bout effect. However, cyclists maintained their cycling completion times during exercise-induced muscle damage (EIMD) in both resistance training bouts, possibly by altering their cycling strategies. Thus, cyclists should consider EIMD symptomatology after resistance training bouts, particularly for cycling-specific technical sessions, regardless of the repeated bout effect.