This study aimed to investigate the effects of percutaneous acupuncture stimulation on the viscoelasticity of human tendon structures during isometric contraction. Nine healthy men participated. The experimental order was pre-test, acupuncture stimulation, and post-test. Real and sham acupuncture applications were used at the stimulus site of the medial gastrocnemius muscle (MG), and a crossover trial was performed on the same subjects at a later date. Before and after acupuncture stimulation, tendon elongation and MG aponeurosis were directly measured by ultrasonography while the subjects performed isometric plantar flexions up to the maximum voluntary contraction (MVC) followed by relaxation. The relationship between the estimated MG muscle force (Fm) and tendon elongation (L) during the ascending phase was fitted to a linear regression, the slope of which was defined as the stiffness of the tendon structures. Additionally, the ratio (%) of the area within the Fm–L loop to the area under the curve during contraction and relaxation was calculated and defined as hysteresis. Stiffness rate of change (RC) in real and sham acupuncture was 137.5±116.5% and 55.0±10.4%, respectively (p<0.05). Thus, real acupuncture demonstrated significantly higher values than sham acupuncture. The hysteresis measurement results in real acupuncture indicated a downward tendency (pre-treatment: 25.6±5.1%, post-treatment: 16.1±13.0%), while sham acupuncture indicated an upward tendency (pre-treatment: 26.5±10.9%, post-treatment: 28.4±6.9%). These results indicated that percutaneous acupuncture stimulation reduces hysteresis, enhances stiffness, and improves the viscoelasticity of tendon structures.