We analyzed stress-strain curves in transverse sections at three different root levels of the cervical, middle, and apical regions of the distal root of the mandibular first molar in hamsters fed a high-carbohydrate (experimental group) or standard (control group) diet for 8 weeks. Each section was loaded in vitro at a rate of 5 mm/min in an extrusive direction. The rupture sites and the structures of birefringent collagen fibers in the periodontal ligament were also analyzed. The maximum shear stress, tangent modulus, and failure strain energy density of the periodontal ligament were significantly greater in the cervical region than in the middle and apical regions in both the control and experimental groups. The maximum shear stress, tangent modulus, and failure strain energy density of the periodontal ligament in the experimental group were significantly lower than those in the control group at the cervical and middle regions. The maximum shear strain was significantly greater in the experimental group than in the control group at the middle and apical regions. Histological sections showed that the periodontal ligament ruptured irregularly in both groups. The collagenous fibers of the ligament in the experimental group appeared to be thinner and less birefringent at the cervical and middle regions of the root. These results suggest that a high-carbohydrate diet differentially affects the collagen fibers in the periodontal ligament as a function of the root level, and that these effects are evident in changes in the mechanical properties of the ligament.