Supramaximal sprint interval training (SIT) provides a potent stimulus for improving maximal aerobic capacity (VO2max), which is a strong marker for both endurance performance and future cardiovascular health and premature mortality. Cycling based SIT typically involves six or more ‘all-out’ 30-s Wingate sprints per training session, yet we have recently demonstrated that similar improvements in VO2max can be achieved with as few as two 20-s sprints (1). This suggests that the volume of sprint exercise has limited influence on subsequent adaptations in VO2max. In this study, we aimed to examine whether a single 20-s ‘all-out’ cycle-sprint per training session can provide a sufficient stimulus for improving VO2max. Thirty sedentary participants (10 men / 20 women; mean±SD age 24±6 y, BMI 22.6±4.0 kg/m2, VO2max 33.2±7.1 mL/kg/min) were randomised to a training group (n=16) or a no-intervention control group (n=14). Training involved three exercise sessions per week for four weeks, consisting of a single 20-s Wingate sprint (no warm-up or cool-down). VO2max was determined prior to training and three days following the final training session. Mean VO2max did not significantly change in the training group (2.15±0.62 vs. 2.22±0.64 L/min) or the control group (2.07±0.69 vs. 2.08±0.68 L/min; effect of time: P=0.17; group x time interaction effect: P=0.26). In conclusion, although we have previously demonstrated that regularly performing two repeated cycle-sprints provides a sufficient training stimulus for a robust increase in VO2max, our present study suggests that this is not the case when training sessions are limited to a single 20-s sprint.