This study tested the utility of optical coherence tomography (OCT)-based indentation to assess mechanical properties of respiratory tissues in disease. Using OCT-based indentation, the elastic modulus of mouse diaphragm was measured from changes in diaphragm thickness in response to an applied force provided by an indenter. We used a transgenic mouse model of chronic lung disease induced by the overexpression of transforming growth factor-alpha (TGF-α), established by the presence of pleural and peribronchial fibrosis and impaired lung mechanics determined by the forced oscillation technique and plethysmography. Diaphragm elastic modulus assessed by OCT-based indentation was reduced by TGF-α at both left and right lateral locations (p <0.05). Diaphragm elastic modulus at left and right lateral locations were correlated within mice (r = 0.67, p <0.01) suggesting that measurements were representative of tissue beyond the indenter field. Co-localised images of diaphragm after TGF-α overexpression revealed a layered fibrotic appearance. Maximum diaphragm force in conventional organ bath studies was also reduced by TGF-α overexpression (p <0.01). Results show that OCT-based indentation provided clear delineation of diseased diaphragm, and together with organ bath assessment, provides new evidence suggesting that TGF-α overexpression produces impairment in diaphragm function and, therefore, an increase in the work of breathing in chronic lung disease.