This study investigated the changes in fascicle length (FL), musculotendinous unit (MTU) stiffness, muscle oxygen saturation (SmO2), and muscular performance following a single bout of a combination of static stretching (SS) or dynamic stretching (DS) with foam rolling (FR), SS only, and eccentric exercise (ECC) only in young men with hamstring tightness. Twenty-five men (20.5 ± 1.5 years) participated in a crossover randomized study of the four conditions (DS+FR, SS+FR, SS, and ECC); each session was spaced seven days apart. FL, MTU stiffness during the straight leg raise (SLR), hamstring SmO2, and isometric and eccentric torque were measured before, immediately and 30 min after exercise. Immediately after exercise, the SLR increased significantly by means of 7.4% (d = 1.07), 6% (d = 1.27), 6% (d = 1.10), and 8% (d = 1.04, all p < 0.001) for DS+FR, SS+FR, ECC, and SS, respectively. FL was longer after exercise under all four conditions (p < 0.05). MTU stiffness decreased after ECC (p = 0.038, d = 0.40). SmO2 tended to decrease for ECC (p > 0.05), but it was increased immediately after those three exercises. Hamstring isometric torque was significantly reduced by an average of 6–9%, but eccentric torque changes varied among conditions. In conclusion, substantial and similar immediate increases in hamstring flexibility, coupled with reduced isometric torque following various exercises, were accompanied by condition-specific alterations in fascicle length, MTU stiffness, or SmO2. These findings provide practical insights for acutely enhancing range of motion in individuals with tight hamstrings.