The enthalpy change (relaxation enthalpy) of the glycerol glass with annealing time was studied using DSC for different annealing temperatures. The investigation of the fictive temperature and glass transition temperature variations with annealing parameters was also carried out. In this study, a well-annealed glass was used as a reference for the determination of relaxation enthalpy. Two enthalpy contributions to relaxation enthalpy were observed. The results were discussed in terms of a multi-step thermodynamic transformation of molecular associations in the glass transition range. A linear relation was also found between relaxation enthalpy and fictive temperature. This well-known conclusion was established from experimental results. Finally, an empirical relation was proposed for the variation of the fictive temperature with annealing parameters.