Inter-structural release (ISR) procedure to skeletal muscles is believed to attenuate adhesion between neighboring muscles and alleviate subjective symptoms, including perceived stiffness and associated pain. However, objective evidence about the effect of ISR on the mechanical properties of the muscles is limited. The purpose of this study was to test the acute effect of ISR on the shear modulus of adjacent muscles. It was hypothesized that ISR applied to the border between synergistic muscles would acutely reduce the resting shear modulus of the involved muscle. To assess the effect of ISR, the shear modulus of the resting peroneus longus (PL) and soleus (SOL) muscles was measured at various ankle joint positions (inversion/eversion) before and after interventions in nine non-symptomatic male individuals (age: 21.9 ± 1.4 years, height: 170 ± 6.6 cm, body mass: 64.3 ± 4.0 kg). Potential muscle activity during the measurement was assessed by recording surface electromyogram from these muscles. The interventions were ISR to the lower leg between the deep fascia of the PL and SOL and sham (massage). As the ankle joint position was passively varied toward inversion, the shear modulus increased substantially in PL and decreased moderately in SOL. Shear modulus of the proximal PL in the most inverted position increased acutely after ISR (by 11.4 ± 5.8%, P < 0.001), but not after the sham, without any change in the amplitude of electromyogram. These results provide objective evidence that the ISR procedure can acutely alter shear modulus, at least in a specific site in a resting muscle.