Background: Prosthetic alignment is a procedure in which a prosthetist finds the most proper position of the prosthetic components according to multi-criteria evaluation of the amputee's gait and standing. Therefore, identifying a set of parameters that describe the relationship between the alignment and the amputee's gait or stance would help the prosthetist to identify a correct prosthetic alignment. Research objective: To identify the effect of prosthetic alignment on muscle activity, comfort, kinetic and kinematic information during gait and standing in transfemoral amputees. Methods: The ethics committee of the University of Antioquia endorsed the experiments. Sixteen transfemoral amputees were recruited. Two prosthetists accompanied the test and performed the prosthesis alignments. Two types of alignments were performed, one nominal and four misalignments. The misalignments were achieved by altering the nominal alignment of the socket and prosthetic foot. Electromyography of the sound limb was recorded, motion capture was performed, and the ground reaction force was recorded during walking and standing. Finally, a survey was applied to evaluate comfort during alignment. Results: The misaligned prostheses produced a more fatiguing gait and greater muscle activity than with the properly aligned prostheses. The amputee increased gait speed and prosthetic limb cadence during use of the misaligned prostheses. In addition, the amputee's weight distribution over the socket was affected, whereby the prosthetic limb showed differences between the vertical and anteroposterior ground reaction force curves for each alignment condition. Prosthetic misalignment resulted in discomfort and increased frustration of the amputee with the prosthesis. They also reported back pain caused by the misalignment, as well as stability problems during walking and standing. Importance: This study presents the biomechanical parameters most affected by prosthetic misalignment in transfemoral amputees, so that prosthetists and engineers can use this information to detect signal patterns and propose new clinical procedures for prosthetic alignment.