Foods meet acid pH during gastric digestion after cooking. An in situ infrared microspectroscopy approach was developed to detect the effects of heat and acid treatments on protein structure separately. Infrared spectra were obtained from meat samples treated with heat and/or acid, and wavenumbers accounting independently for the treatments were extracted by principal component regression. Extreme-acid treatment (pHinitial 2.0) was well predicted (0.5% error) by a simple ratio of as-observed spectral intensities at 1211 and 1396 cm-1, reflecting a perturbation in the vibration of amino acid residues (phenylalanine, tyrosine and aspartic acid) by protein unfolding and protonation. Using the imaging mode of an IR microscope, meat protein acidification was evidenced with high spatial resolution. The heat effect was well discriminated from the acid effect by the ratio of as-observed intensities at 1666 and 1697 cm-1 (0.9% error), indicating content of aggregated β-sheets relative to α-helix structure.