The aim of this study was to determine differences in glycolytic metabolite concentrations and work output in response to an all-out interval training session in 23 cyclists with at least 2 years of interval training experience (E) and those inexperienced (IE) in this form of training. The intervention involved subsequent sets of maximal intensity exercise on a cycle ergometer. Each set comprised four 30 s repetitions interspersed with 90 s recovery periods; sets were repeated when blood pH returned to 7.3. Measurements of post-exercise hydrogen (H+) and lactate ion (LA-) concentrations and work output were taken. The experienced cyclists performed significantly more sets of maximal efforts than the inexperienced athletes (5.8 ± 1.2 vs. 4.3 ± 0.9 sets, respectively). Work output decreased in each subsequent set in the IE group and only in the last set in the E group. Distribution of power output changed only in the E group; power decreased in the initial repetitions of set only to increase in the final repetitions. H+ concentration decreased in the third, penultimate, and last sets in the E group and in each subsequent set in the IE group. LA- decreased in the last set in both groups. In conclusion, the experienced cyclists were able to repeatedly induce elevated levels of lactic acidosis. Power output distribution changed with decreased acid–base imbalance. In this way, this group could compensate for a decreased anaerobic metabolism. The above factors allowed cyclists experienced in interval training to perform more sets of maximal exercise without a decrease in power output compared with inexperienced cyclists.