This study aimed to assess the feasibility of using quantitative shear wave elastography (SWE) to assess the stiffness (Young's modulus) of Achilles tendon and to investigate the effect of long-term weight-bearing exercise on the stiffness of human Achilles tendon by comparing the frequent and infrequent exercise groups.Case-control study.A total of 36 healthy subjects aged 19–25 were recruited. Subjects were categorized into frequent and infrequent exercise groups dependent on their level of lower limb weight bearing exercise. B-mode and shear-wave ultrasound examination of Achilles tendon were performed. Measurements of the Achilles tendon stiffness were conducted with the foot position standardized by an ankle fixer. To evaluate inter and intra-operator reliability of Young's modulus measurements, each subject was scanned by three operators and each operator scanned the subject three times.The intra-operator reliability of Young's modulus measurements ranged between 0.803 and 0.845. The inter-operator measurement reliability was 0.585. Result showed that Achilles tendon on non-dominant ankle in frequent-exercisers (median: 320.1kPa) was significantly stiffer than that in infrequent-exercisers (median: 296kPa) (p<0.05), whereas there was no significant difference in Achilles tendon stiffness on dominant ankle between the two groups (p>0.05).Shear wave elastography is feasible for assessing the Achilles tendon's stiffness in vivo. The stiffness of Achilles tendon of frequent-exercisers was significantly higher than that of infrequent-exercisers on non-dominant ankle but not on dominant ankle. With the use of an ankle fixer, there was high repeatability and moderate reproducibility in SWE measurement of Achilles tendon stiffness.