The aims of this study were to determine the reliability of an intermittent-sprint cycling protocol and to determine the efficacy of one practice session on main trials. Eleven men, moderately trained team-sport athletes, completed three visits to the laboratory involving a graded-exercise test and practice session and two trials of a cycling intermittent-sprint Protocol separated by three days. Data for practice and main trials were analysed using typical error of measurement, intra-class correlation and least-products regression to determine reliability. Typical error of measurement (expressed as a coefficient of variation) and intra-class correlation for peak power output from all 20 sprints for trial 1 and trial 2 were 2.9 ± 12.8% (95% confidence interval: 2.0-5.0%) and 0.96 (95% confidence interval: 0.85-0.99), respectively. Typical errors of measurement and intra-class correlation for mean power output for all 20 sprints for trials 1 and 2 were 4.2 ± 11.9% (95% confidence interval: 2.9-7.4%) and 0.90 (95% confidence interval: 0.66-0.97), respectively. The results suggest that peak power output provides a more reliable measure than mean power output. The Cycling Intermittent-Sprint Protocol provides reliable measures of intermittent-sprint performance.