Human serum albumin (HSA) is the most abundant protein in human plasma. Most protein dynamics studies of HSA have been performed above the hyperthermia temperature (>42 °C), so information on the dynamics under hypothermic conditions (<35 °C) is lacking. In this work, a tryptophan-based fluorescence temperature jump system was employed to investigate the thermally-induced dynamic process of HSA at a physiological concentration of ca. 45 mg mL-1 and pH = ca. 7 upon an instantaneous temperature increase from 25 °C to 30-43 °C. The observed kinetics manifested a three-state consecutive feature, . Upon analysis with the Arrhenius model, the rate coefficients k1 and k2 manifested piecewise temperature dependence, and the turning-point temperature was found to be ca. 34 °C, coinciding with the upper bound of hypothermic temperature. Meanwhile, the corresponding activation energies of the transitions at 34-43 °C were lower than those at 30-34 °C, suggesting that protein conformational adjustments at 34-43 °C were more feasible than those at hypothermic temperatures. These observations provided a fresh viewpoint on the relationship between the energetics of protein dynamics and the apparent functioning of a given protein at the molecular level.