Although the overall bulk properties of the Achilles tendon have been measured, there is little information detailing the properties of individual fascicles or their interactions. The knowledge of biomechanical properties at the fascicle-scale is critical in understanding the biomechanical behavior of tendons and for the construction of accurate and detailed computational models. Seven tissue samples harvested from four freshly thawed human (all male) tendons, each sample having four to six fascicles, were tested in uniaxial tension. A sequential sectioning protocol was used to isolate interaction effects between adjacent fascicles and to obtain the loading response for a single fascicle. The specimen deformation was measured directly using a novel polarized light imaging system with digital image correlation (DIC) for marker-free deformation measurement. The modulus of the single fascicle was significantly higher compared with the intact fascicle group (single: 226 MPa (SD 179), group: 68 MPa (SD 33)). The interaction effect between the adjacent fascicles was less than 10% of the applied load and evidence of sub- and postfailure fascicle sliding was clearly visible. The DIC direct deformation measurements revealed that the modulus of single fascicles could be as much as three to four times the intact specimen. The consistently higher moduli values of the single (strongest) fascicle indicate that the overall response of the tendon may be dominated by a subset of “strongest” fascicles. Also, fascicle-to-fascicle interactions were small, which was of the overall response. This knowledge is useful for developing computational models representing single fascicle and/or fascicle group mechanical behavior and provides valuable insights into fascicle-scale Achilles tendon material properties.