Increased muscle stiffness of the pectoralis minor (PMi) could deteriorate shoulder function. Stretching is useful for maintaining and improving muscle stiffness in rehabilitation and sport practice. However, the acute and prolonged effect of stretching on the PMi muscle stiffness is unclear due to limited methodology for assessing individual muscle stiffness. Using shear wave elastography, we explored the responses of shear modulus to stretching in the PMi over time. The first experiment (n = 20) aimed to clarify the acute change in the shear modulus during stretching. The shear modulus was measured at intervals of 30 s × 10 sets. The second experiment (n = 16) aimed to observe and compare the prolonged effect of different durations of stretching on the shear modulus. Short and long stretching duration groups underwent 30s × 1 set and 30s × 10 sets, respectively. The assessments of shear modulus were conducted before, immediately after, and at 5, 10, and 15 min post-stretching. In experiment I, the shear modulus decreased immediately after a bout (30 s) of stretching (p < 0.001, change: -2.3 kPa, effect size: r = 0.72) and further decreased after 3 repetitions (i.e., 90 s) of stretching (p = 0.03, change: -1.0 kPa, effect size: r = 0.53). In experiment II, the change in the shear modulus after stretching was greater in the long duration group than in the short duration group (p = 0.013, group mean difference: -2.5 kPa, partial η² = 0.36). The shear modulus of PMi decreased immediately after stretching, and stretching for a long duration was promising to maintain the decreased shear modulus. The acute and prolonged effects on the PMi shear modulus provide information relevant to minimum and persistent stretching time in rehabilitation and sport practice.

• Mechanical responses of the pectoralis minor muscle to stretching over time were examined.

• Shear modulus was used as a surrogate of muscle stiffness.

• Shear modulus of the pectoralis minor decreased after a bout (= 30s) of stretching.

• Shear modulus further decreased after 3set (= 90s) of stretching.

• Different prolonged effect resulted from the different stretching durations.