FT-NIR spectra were measured for human serum albumin (HSA) in aqueous solutions with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 wt % over a temperature range of 45−80 °C. Concentration-perturbed two-dimensional (2D) correlation spectra were calculated for the spectra in the 7500−5500 cm-1 and 4900−4200 cm-1 regions at different temperatures. To investigate temperature-induced changes in the secondary structure and hydration, power spectra and slice spectra were calculated from the synchronous and asynchronous spectra, respectively. In the power spectra, a band near 4600 cm-1 due to the combination mode of amide B and amide II (amide B/II) shows an abrupt shift by 5 cm-1 between 58 and 60 °C, indicating that the secondary structure of HSA changes suddenly near 60 °C. Both the power and slice spectra in the 7500−5500 cm-1 region provide explicit evidence that the hydration changes markedly near 60 °C. A comparison of the temperature-dependent frequency shifts between the band near 4600 cm-1 due to amide ...