The aim of this study was to investigate the effects of the Nordic hamstring exercise on the biceps femoris long head (BFlh), biceps femoris short head (BFsh), semitendinosus (SMT), and semimembranosus (SMM) muscles. The Nordic hamstring strengthening exercise has been widely used in injury prevention, yet not much is known about the site-specific activation of this exercise on different muscles of the thigh. Eight male national-level referees were assigned to a Nordic hamstring exercise protocol (5 sets of 8 repetitions). Magnetic resonance imaging (MRI) of the subjects' thighs was performed before, within 3 minutes after, and repeated again 72 hours after the exercise intervention. Fifteen axial scans of the thigh interspaced by a distance of 1 of 15 right femur length were obtained from the level of 1 of 15 Lf to 15 of 15 Lf. The MRI data were analyzed for signal intensity changes. After 72 hours, significant changes in transverse (spin-spin) relaxation time signal intensity and cross-sectional area were maintained distally at BFsh cranial portion and concretely at the nondominant limb, whereas no significant changes were observed in transverse (spin-spin) relaxation time signal intensity at BFlh, SMM, or SMT. This study demonstrated that the Nordic hamstring exercise did not result in a uniform response (training stimulus) neither interhamstring (dominant vs. nondominant) nor intrahamstring muscles (same leg) and was better suited for loading proximal BFsh.