Sex differences in the mechanical properties of different musculoskeletal tissues and their impact on tendon function and disease are becoming increasingly recognized. Tendon mechanical properties are influenced by the presence or absence of sex hormones and these effects appear to be tendon- or ligament-specific. The objective of this study was to determine how sex and hormone differences in rats affect supraspinatus tendon and muscle properties. We hypothesized that male supraspinatus tendons would have increased cross-sectional area but no differences in tendon material properties or muscle composition when compared to supraspinatus tendons from female or ovariectomized (OVX) female rats. Uninjured supraspinatus tendons and muscles from male, female, and OVX female rats were collected and mechanical and histological properties were determined. Our analysis demonstrated decreased dynamic modulus and increased hysteresis and cross-sectional area in male tendons. We found that male tendons exhibited decreased dynamic modulus (during low strain frequency sweep and high strain fatigue loading), increased hysteresis, and increased cross-sectional area compared to female and OVX female tendons. Despite robust mechanical differences, tendon cell density and shape, and muscle composition remained unchanged between groups. Interestingly, these differences were unique compared to previously reported sex differences in rat Achilles tendons, which further supports the concept that the effect of sex on tendon varies anatomically. These differences may partially provide a mechanistic explanation for the increased rate of acute supraspinatus tendon ruptures seen in young males.