Nanofiber yarns with twisted and continuous structures have potential applications in fabrication of complicated structures such as surgical suture yarns, artificial blood vessels, and tissue scaffolds. The objective of this article is to characterize the tensile fatigue behavior of continuous Polyamide 66 (PA66) nanofiber yarns produced by electrospinning with three different twist levels. Morphology and tensile properties of yarns were obtained under static tensile loading and after fatigue loading. Results showed that tensile properties and yarn diameter were dependent on the twist level. Yarns had nonlinear time independent stress-strain behavior under the monotonic loading rates between 10 and 50 mm/min. Applying cyclic loading also positively affected the tensile properties of nanofiber yarns and changed their stress-strain behavior. Fatigue loading increased the crystallinity and alignment of nanofibers within the yarn structure, which could be interpreted as improved tensile strength and elastic modulus. (C) 2014 Society of Plastics Engineers