Abstract Tendons are composed of type I collagen fibrils and specialized extracellular matrix (ECM) proteins that are hierarchically organized to transmit forces from muscle to bone. Reliable and affordable models of human tendon constructs are requested to serve surgical treatment after tendon injury and in vitro pharmacological testing. Here, using human adipose stromal cells embedded in a 3D type-I collagen matrix and submitted to static uniaxial geometrical constraint, we generated human tendon constructs and assessed their mechanical properties and gene expression for up to 21 days in culture. The analysis of visco-elastic properties by nano-indentation indicated an increase in tissue stiffness (from about 1kPa at Day 0 to 10 kPa at Day 21), concomitant with a reduced percentage of stress relaxation, indicative of more solid-like mechanical properties. These changes in mechanical properties were associated with a reorganisation of ECM and the emergence of two cell populations, with one cell population organized as a ring in tissue periphery. In addition to the activation of the well-established tendon differentiation genetic program, assessed by the expression of SCX , MKX , TNMD and COL1A1 , we validated the expression of a novel tendon marker, TM4SF1 and other components of the ECM such as COL6A3 , COL14A1 , DPT , DCN , POSTN and THBS2 , which were previously identified in transcriptomic analyses of embryonic mouse limb tendons. In summary, this 3D culture system, which recapitulates most stages of tendon development, represent a potential route for human tendon regeneration.