The effect of fire on the Simon Pereyns’s Virgen del Perdon was studied. This panel painting from sixteenth century was damaged in the 1967 fire at Mexico City Cathedral. The physicochemical interpretation of alterations due to the raise of temperature is based on experimental reproduction of the painting structure, starting from the support, which underwent controlled temperature increase. This research arise from the understanding of Virgen del Perdon manufacture technique, the revision of historical sources and art treatises, and the comparative scientific analysis between original materials and experimental reproductions. The study was developed from an interdisciplinary approach with biological identification of organic materials and characterization of inorganic materials through optical microscopy, XRD and SEM-EDS.