Effective cycle training for triathlon is a challenge for coaches. We compared the effects of two variants of cycle high-intensity interval training (HIT) on triathlon-specific cycling and running. Fourteen moderately-trained male triathletes (O2peak 58.7±8.1 mL kg− 1 min− 1; mean±SD) completed on separate occasions a maximal incremental test (O2peak and maximal aerobic power), 16× 20 s cycle sprints and a 1-h triathlon-specific cycle followed immediately by a 5 km run time trial. Participants were then pair-matched and assigned randomly to either a long high-intensity interval training (LONG)(6–8× 5 min efforts) or short high-intensity interval training (SHORT)(9–11× 10, 20 and 40 s efforts) HIT cycle training intervention. Six training sessions were completed over 3 weeks before participants repeated the baseline testing. Both groups had an∼ 7% increase in O2peak (SHORT 7.3%,±4.6%; mean,±90% confidence limits; LONG 7.5%,±1.7%). There was a moderate improvement in mean power for both the SHORT (10.3%,±4.4%) and LONG (10.7%,±6.8%) groups during the last eight 20-s sprints. There was a small to moderate decrease in heart rate, blood lactate and perceived exertion in both groups during the 1-h triathlon-specific cycling but only the LONG group had a substantial decrease in the subsequent 5-km run time (64,±59 s). Moderately-trained triathletes should use both short and long high-intensity intervals to improve cycling physiology and performance. Longer 5-min intervals on the bike are more likely to benefit 5 km running performance.