Background: Collagen, the most abundant protein in the animal kingdom, represents a promising biomaterial for regenerative medicine applications due to its structural diversity and self-assembling complexity. Despite collagen’s widely known structural and functional features, the thermodynamics behind its fibrillogenic self-assembling process is still to be fully understood. In this work we report on a series of spectroscopic, mechanical, morphological and thermodynamic characterizations of high purity type I collagen (with a D-pattern of 65 nm) extracted from Wistar Hannover rat tail. Our herein reported results can be of help to elucidate differences in self-assembly states of proteins using ITC to improve the design of energy responsive and dynamic materials for applications in tissue engineering and regenerative medicine. Methods: Herein we report the systematic study on the self-assembling fibrillogenesis mechanism of type I collagen, we provide morphological and thermodynamic evidence associated to different self-assembly events using ITC titrations. We provide thorough characterization of the effect of pH, effect of salts and protein conformation on self-assembled collagen samples via several complementary biophysical techniques, including circular dichroism (CD), Fourier Transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), dynamic mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA). Results: Emphasis was made on the use of isothermal titration calorimetry (ITC) for the thermodynamic monitoring of fibrillogenesis stages of the protein. An overall self-assembly enthalpy value of 3.27 ± 0.85 J/mol was found. Different stages of the self-assembly mechanism were identified, initial stages take place at pH values lower than the protein isoelectric point (pI), however, higher energy release events were recorded at collagen’s pI. Denatured collagen employed as a control exhibited higher energy absorption at its pI, suggesting different energy exchange mechanisms as a consequence of different aggregation routes.