We have developed an in vitro model system to investigate the relationships between mechanical unloading and tendon matrix remodeling. Remodeling was characterized by changes in the functional and structural characteristics of rat tail tendon fascicles (RTTF) subjected to no load conditions for 1 week in vitro. We hypothesized that the absence of load will: (I) maintain cross-sectional area (CSA), with decreased elastic modulus and increased stress-relaxation; (II) cause an increase in denatured collagen and a decrease in water and total glycosaminoglycan (GAG) content. Fascicles cultured under a nominal static stress were used as control for culture conditions effects. Unloading resulted in a decrease of approximately 23% in the elastic modulus of cultured fascicles, consistent with previous stress-deprivation studies. Contrary to our hypothesis, a nominal static stress caused an increase in elastic modulus ( approximately 30%) and a significant decrease in stress-relaxation when compared to fresh fascicles at 1% strain. Mechanical changes were associated with changes in the GAG content of the fascicles, but not their CSA, water, or collagen content. Furthermore, we did not find evidence of measurable denatured collagen in the cultured fascicles. Together these results suggest a role for GAG but not collagen or water in the elastic and viscoelastic changes measured in tendon fascicles cultured for 1 week under altered load conditions.