The purpose of this study was to investigate the acute physiological responses of interval protocols using the minimal power output (MAP) that elicits peak oxygen uptake (V[Combining Dot Above]O2peak) as exercise intensity and different durations of work intervals during intermittent cycling. In randomized order, 13 well-trained male cyclists (V[Combining Dot Above]O2peak = 67 ± 6 ml·kg·min) performed 3 different interval protocols to exhaustion. Time to exhaustion and time ≥ 90% of V[Combining Dot Above]O2peak were measured with MAP as exercise intensity, and work duration of the intervals equals either 80% of Tmax, 50% of Tmax, or 30 seconds with recovery period being 50% of the work duration at intensity equal to 50% of MAP. The major findings were that the interval protocol using 30-second work periods induced longer time ≥90% of V[Combining Dot Above]O2peak and longer work duration at MAP intensity than the interval protocols using work periods of 50% of Tmax or 80% of Tmax (p ≤ 0.05). There was no difference between the protocols using work periods of 50% of Tmax or 80% of Tmax. In conclusion, the present study suggests that the 30-second work interval protocol acutely induces a larger exercise stimulus in well-trained cyclists than the protocols using work periods of 50% of Tmax or 80% of Tmax. The practical application of the present findings is that fixed 30-second work intervals can be used to optimize training time at MAP and time ≥90% of V[Combining Dot Above]O2peak in well-trained cyclists using MAP exercise intensity and a 2:1 work:recovery ratio.