This study investigated the biomechanical effects of thenar muscles (abductor pollicis brevis (APB), superficial head of flexor pollicis brevis (sFPB), opponens pollicis (OPP)) on the transverse carpal ligament formed carpal arch under force application by individual or combined muscles (APB, sFPB, OPP, APB-sFPB, sFPB-OPP, APB-OPP, and APB-sFPB-OPP). In ten cadaveric hands, thenar muscles were loaded under 15% of their respective maximal force capacity, and ultrasound images of the cross section of the distal carpal tunnel were collected for morphometric analyses of the carpal arch. The carpal arch height and area were significantly dependent on the loading condition (p < 0.01), muscle combination (p < 0.05), and their interaction (p < 0.01). The changes to arch height and area were significantly dependent on the muscle combinations (p = 0.001 and p < 0.001, respectively). The arch height and area increased under the loading combinations of APB, OPP, APB-sFPB, APB-OPP, or APB-sFPB-OPP (p < 0.05), but not under the combinations of sFPB (p = 0.893) or sFPB-OPP (p = 0.338). The carpal arch change under the APB-sFPB-OPP or APB-OPP loading was greater than that under the loading of APB-sFPB (p < 0.001). This study demonstrated that thenar muscle forces exert biomechanical effects on the transverse carpal ligament to increase carpal arch height and area, and these increases were different for individual muscles and their combinations.