The mechanical response of a biological material to applied forces reflects deformation mechanisms occurring within a hierarchical architecture extending over several distinct length scales. Characterizing and in turn predicting the behaviour of such a material requires an understanding of the mechanical properties of the substructures within the hierarchy, the interaction between the substructures, and the relative influence of each substructure on the overall behaviour. While significant progress has been made in mechanical testing of micrometre to millimetre sized biological specimens, quantitative reproducible experimental techniques for making mechanical measurements on specimens with characteristic dimensions in the smaller range of 10–1000 nm are lacking. Filling this void in experimentation is a necessary step towards the development of realistic multiscale computational models useful to predict and mitigate the risk of bone fracture, design improved synthetic replacements for bones, tendons and ligaments, and engineer bioinspired efficient and environmentally friendly structures. Here, we describe a microelectromechanical systems device for directly measuring the tensile strength, stiffness and fatigue behaviour of nanoscale fibres. We used the device to obtain the first stress–strain curve of an isolated collagen fibril producing the modulus and some fatigue properties of this soft nanofibril.