In vivo tests and design of experiments were carried out to determine the influence of experimental factors on mechanical response of soft tissue. This study proposes an inverse method to obtain constitutive equations of a multilayer biological model (cutis, hypodermis, and muscle) through the use of indentation tests in combination with a finite element method. An experimental design was developed to determine the incidence of the following factors on the mechanical behavior of skin: room temperature (A), indentation velocity (B), indenter temperature (C), pump pressure (D), and muscle activation (E). The factor D and factor interactions ADE, CDE, and ACDE were statistically significant with respect to skin mechanical response. For each combination of these factors, one constitutive equation was established. Sixteen constitutive equations were obtained for use in further numerical models in which experimental conditions must be taken into account. Therefore, it can be concluded that there is not a unique equation able to represent the mechanical properties of skin under all the conditions considered in this study.