Testing patients with a uni-lateral leg amputation using a graded one-legged peak exercise test (GOPET) poses difficulties. The smaller muscle mass results in increased intramuscular tension, thereby restricting blood flow to the active muscle. This ultimately leads to enhanced local fatigue in the active muscles. This is aggravated when vascular problems are present, such as often seen in persons who underwent amputation. This study investigated the influence of a discontinuous protocol when performing a GOPET in healthy participants. It is hypothesized that the resting phase following the exercise phase will restore blood flow, postponing local fatigue resulting in a higher measured aerobic capacity. Eight healthy participants performed three GOPET's: a continuous protocol (CON) and two discontinuous protocols in which an exercise phase of 60 (DIS60) or 90 s (DIS90) was followed by a 30 s rest period. No difference in aerobic capacity and peak heart rate were found among the three protocols. Peak workload and exercise duration were significantly higher in both discontinuous protocols compared to the continuous protocol. Subjectively, participants experienced the DIS protocols as more comfortable. A discontinuous protocol elicits similar aerobic capacities and peak heart rates, but higher peak workloads and lower discomfort, compared to a continuous protocol. Given the subjective preference, a discontinuous protocol may be advantageous over a continuous protocol, which may especially apply for patients with an amputation due to vascular problems.