Mechanical stress plays a significant role in modulating cell behaviour and has driven the development of mechanical bioreactors for tissue engineering applications. Our aim was to investigate the effect of cyclic mechanical stimulation on human tenocytes seeded onto hyaluronic acid based scaffold. Human tenocytes were isolated from tendon samples obtained from hand lesions. Tenocytes were expanded in vitro and then seeded onto hyaluronic acid based scaffold. Tissue constructs were placed into a bioreactor, devised by the authors, able to reproduce a physiological mechanical traction. Controls were left untensioned. Histological, immunohistochemical, biomolecular, transmission and miscoscopic electron analyses were used to evaluate the results after 7, 14, and 21 days. Tenocytes adhered and proliferated within the biomaterial and produced main components of the extracellular matrix. Microscopically, cyclically tensioned samples showed parallel orientation of collagen fibers and spindle- shaped cell nuclei mimicking the morphology of normal tendons. Mechanostimulation resulted in significantly stronger and stiffer constructs compared to untensioned samples. Higher expression of matrix proteins such as collagen I and adhesion proteins such as integrin 1 and scleraxis (tendon specific markers) were found in cyclically tensioned samples. Human tenocytes were able to develop a structure similar to normal tendon if mechanical stress were applied in vitro. Mechanical traction was essential to maintain cellular phenotype, and enhanced cell proliferation and tenocytes longitudinal alignment. Duration, frequencies, and amplitude of loading directly influence cell behaviour in vitro. Understanding the physiological window for these parameters represents future challenges of research in the field of tendon tissue engineering.