Objectives: This study investigated the acute effects of dynamic stretch (DS) duration on the muscle stiffness of the ankle plantar flexor using shear wave ultrasound elastography. Methods: Eighteen healthy young participants were enrolled in this study. DS with one set (DS1) or four sets (DS4) of 30 s each was performed randomly. Shear wave velocity in the medial gastrocnemius (MG) was measured before and after DS to assess muscle stiffness of the MG. Results: Two-way repeated-measures analysis of variance (condition × time) showed a significant interaction with the shear wave velocity (p = 0.02). Shear wave velocity significantly decreased after the DS4 than before (before: 3.09±0.59 m/s; after: 2.86±0.43 m/s). However, no significant differences were observed in shear wave velocity between before and after DS1 (before: 2.96±0.56 m/s; after: 3.19±0.56 m/s). There were no significant differences in shear wave velocity at baseline condition. After the intervention, significantly lower shear wave velocity was observed in DS4 than in DS1. Conclusions: The results of this study demonstrate that DS with four sets of 30 s effectively decreased the muscle stiffness of the MG.