The aim of this study was to investigate the feasibility, test-retest reliability, and construct validity of the 2-min step test (2MST) in measuring aerobic exercise capacity and functional endurance in participants with anterior cruciate ligament reconstruction (ACLR). Fifty participants with quadriceps tendon autograft, bone-patellar tendon-bone graft, or hamstring autograft were included. Feasibility was assessed by the time and support required to complete the measures and the feedback from participants, including their satisfaction ratings. Test-retest reliability was assessed using the intraclass correlation coefficients (ICC2,1), a Bland-Altman plot with 95% limits of agreement (LoA), SEM, SEM%, and minimum detectable change (MDC95). The participants were assessed with the visual analogue scale (VAS), Lysholm Knee Scoring Scale, Knee Injury and Osteoarthritis Outcome Score (KOOS), stair climbing test (SCT), and 6-min walk test to assess construct validity. The test took less than 5 min to describe and perform, and participants required minimal verbal support. The satisfaction rate was notably high. The ICC2,1 was 0.98 (0.96-0.99). SEM and MDC95 were 2.96 and 8.20 (2.41-14.00), respectively. The SEM% of 2.99 was considered to be at a very good level. The Bland-Altman plot illustrates that the 2MST scores had a 95% LoA ranging from -21.52 to 2.68 steps. The SCT score and VAS rest score were found to be associated with the 2MST performance, explaining 42.5% of the variance. The 2MST is a feasible, reliable, and valid test to measure aerobic exercise capacity and functional endurance in participants with ACLR.