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Transtibial amputees may experience decreased quality of life due to increased risk of knee joint osteoarthritis (OA). No prior studies have compared knee joint biomechanics for the same group of transtibial amputees in gait, cycling, and elliptical training. Thus, the goal of this study was to identify preferred exercises for transtibial amputees in the context of reducing risk of knee OA. The hypotheses were: 1) knee biomechanics would differ due to participant status (amputee, control), exercise, and leg type (intact, residual) and 2) gait kinematic parameters would differ due to participant status and leg type. Ten unilateral transtibial amputee and ten control participants performed exercises while kinematic and kinetic data were collected. Two-factor repeated measures analysis of variance with post-hoc Tukey tests and non-parametric equivalents were performed to determine significance. Maximum knee compressive force, extension torque, and abduction torque were lowest in cycling and highest in gait regardless of participant type. Amputee maximum knee extension torque was higher in the intact vs. residual knee in gait. Amputee maximum knee flexion angle was higher in the residual vs. intact knee in gait and elliptical. Gait midstance knee flexion angle timing was asymmetrical for amputees and knee angle was lower in the amputee residual vs. control non-dominant knees. The results suggest that cycling, and likely other non-weight bearing exercises, may be preferred exercises for amputees due to significant reductions in biomechanical asymmetries and joint loads., Competing Interests: A. Matt Robinson declares an author commercial affiliation to Hanger Clinic. Mr. Robinson is a licensed Hanger Clinic prosthetist and contributed to study design and implementation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.