Tendinopathy is a frequent and unresolved clinical problem, associated with pain and impaired mobility, having a strong impact on the patient’s life. While a healthy tendon consists of highly aligned collagen, with aligned spindle-shaped tenocytes (the main cell type in tendon), a tendinopathic tendon is characterized by a disorganized matrix, containing stellate-shaped cells. Furthermore, an increase of matrix proteins including collagen type III and fibronectin are associated with tendinopathic tendon. As cell behavior depends on both physical and biochemical stimuli, the changes in the extracellular matrix (ECM) during tendinopathy are a common target for developing treatment strategies. Understanding how both structural and compositional changes affect the tenocyte’s behavior and thereby tenogenic phenotype is essential in identifying more specifically targeted treatment options. Previous studies established a relation between topography-induced tenocyte elongation and phenotype using scleraxis expression as a marker. However, compositional effects, and thereby specific integrin recruitment, have not yet been investigated. Therefore, this study focused on the question whether variation in substrate topography and composition induced by a self-agglomerated collagen type I pattern affect scleraxis expression in tenocytes. Concentric rings in this pattern that drive cells into an elongated shape appeared to have collagen orientations in various directions. No significant differences in scleraxis expression were found between elongated cells and spread cells on the pattern, which indicates that elongated cells do not necessarily have an increased scleraxis expression compared to round cells. When coating the pattern with proteins, fibronectin or collagen type III, a decrease in both scleraxis and integrin α5 expression was observed in tenocytes on coated fibronectin compared to uncoated collagen type I, while no change in scleraxis expression was observed between tenocytes on collagen type III and collagen type I. This underlines the effect of biochemical changes inducing tenocyte phenotype, as well as the potential relation between integrin and scleraxis expression. Taken together, it is suggested that both structural and compositional changes in tendinopathy affect tenocyte phenotype. Hereby, the effect of cell elongation on tenocyte phenotype is affected when biochemical cues are present. In the development of treatment strategies to promote healthy tendon cell behavior, it is therefore suggested that cell elongation induced by an anisotropic matrix is desired, in combination with a change in the proteins present in the ECM, including less fibronectin.