Hierarchical collagen fibers are the primary source of strength in tendons and ligaments; however, these fibers largely do not regenerate after injury or with repair, resulting in limited treatment options. We previously developed a static culture system that guides ACL fibroblasts to produce native-sized fibers and early fascicles by 6 weeks. These constructs are promising ligament replacements, but further maturation is needed. Mechanical cues are critical for development in vivo and in engineered tissues; however, the effect on larger fiber and fascicle formation is largely unknown. Our objective was to investigate whether intermittent cyclic stretch, mimicking rapid muscle activity, drives further maturation in our system to create stronger engineered replacements and to explore whether cyclic loading has differential effects on cells at different degrees of collagen organization to better inform engineered tissue maturation protocols. Constructs were loaded with an established intermittent cyclic loading regime at 5 or 10 % strain for up to 6 weeks and compared to static controls. Cyclic loading drove cells to increase hierarchical collagen organization, collagen crimp, and tissue tensile properties, ultimately producing constructs that matched or exceeded immature ACL properties. Further, the effect of loading on cells varied depending on degree of organization. Specifically, 10 % load drove early improvements in tensile properties and composition, while 5 % load was more beneficial later in culture, suggesting a shift in mechanotransduction. This study provides new insight into how cyclic loading affects cell-driven hierarchical fiber formation and maturation, which will help to develop better rehabilitation protocols and engineer stronger replacements. Statement of significanceCollagen fibers are the primary source of strength and function in tendons and ligaments throughout the body. These fibers have limited regenerate after injury, with repair, and in engineered replacements, reducing treatment options. Cyclic load has been shown to improve fibril level alignment, but its effect at the larger fiber and fascicle length-scale is largely unknown. Here, we demonstrate intermittent cyclic loading increases cell-driven hierarchical fiber formation and tissue mechanics, producing engineered replacements with similar organization and mechanics as immature ACLs. This study provides new insight into how cyclic loading affects cell-driven fiber maturation. A better understanding of how mechanical cues regulate fiber formation will help to develop better engineered replacements and rehabilitation protocols to drive repair after injury.