Tissue engineering is aimed at the fabrication of autologous cardiovascular implants, for example, heart valves or vascular grafts. To date, the mechanical characterization of tissue-engineered vascular grafts (TEVGs) has focused mainly on the material's strength and not on the deformation behavior. A total of 31 samples obtained from 3 mature grafts (out of the cells of a single donor) were tested in uniaxial stress and uniaxial strain configurations to characterize their stiffness under uniaxial and biaxial stress states, respectively. Corresponding measurements were carried out on samples of an ovine artery. A physiological stiffness parameter was defined for data analysis and the uniaxial and multiaxial response compared, also in terms of anisotropy. The tension-strain curve of uniaxial stress tests is highly nonlinear, whereas the results show a more gradual deformation response of the material under a uniaxial strain configuration, which better represents the physiological state of biaxial stress. Stiffness parameters and anisotropy factors are significantly influenced by the selection of the testing configuration. Tangent stiffness of a TEVG at physiological loading conditions is significantly (p<0.05) higher for uniaxial stress as compared to uniaxial strain. The same is observed for the ovine tissue. The anisotropy of the scaffold is shown to partially transfer to the mature TEVG. The results of this study show that for a TEVG characterization, a physiological biaxial testing configuration should be preferred to the commonly used uniaxial stress.