Higher stiffness of the Achilles tendon (AT) and gastrocnemius have been found to be risk factors associated with Achilles tendinitis. Static stretching (SS) is one intervention that has been investigated to improve the flexibility and therefore reduce injury risk. Previous studies have investigated the acute effect of SS on one region for AT and gastrocnemius morphology and stiffness; however, there is a lack of knowledge about the effect of SS on other regions of the AT and gastrocnemius (e.g., proximal vs. distal, within gastrocnemius). The aims of the present study were: (1) to investigate the acute effects of SS on the shear modulus of the medial gastrocnemius muscles (MG) and lateral gastrocnemius muscles (LG) and AT for different regions; (2) to examine the differences in range of motion (ROM) before and after SS; and (3) to investigate the change of thickness of AT and fascicle length of MG and LG before and after SS. The stiffness of AT and the gastrocnemius, fascicle length of the muscles, thickness of the AT, and maximal ankle joint dorsiflexion angle were measured in thirty healthy subjects (15 males, 15 females) before(pre) and immediately after (post) 5-minute SS. Stretching effects are not homogeneous among different regions. After SS administration, the proximal, middle, and distal regions of MG stiffness decreased by 34.12%, 22.45%, and 25.27%, respectively (p = 0.000), and LG stiffness decreased by 37.71%, 30.47%, and 22.13%, respectively (p = 0.000), whereas AT stiffness increased by 25.73%, 17.01%, and 19.53%, respectively (p= 0.000). ROM of ankle joint increased by 8.02% (p=0.00). Nevertheless, there were no changes in the thickness of AT and fascicle length of the gastrocnemius. These results suggest that non-uniform behaviour is consistently present within the gastrocnemius and AT, and the gastrocnemius heterogeneity is reduced after SS. The stretching maneuver could be effective to increase the flexibility.