There exists a significant correlation between the microstructural evolution and the mechanical properties of fibers during repeated loading and unloading cycles. Nevertheless, the influence of deformation and the duration of intervals on the structural and tensile behavior of spider silk after repeated stretching at a given strain value has been rarely reported, with the exception of studies focusing on the major ampullate gland silk (Mas) of the spider. In order to investigate the effects of repeated stretching on the structural and mechanical behavior of spider tubular gland silk (Tus), the tensile properties and the changes in semiquantitative protein secondary structure of