Four panel paintings depicting episodes related to the birth of Christ and attributed to the Portuguese mannerist painter Francisco João (doc. 1558–1585) were found to exhibit a muted palette that had no correspondence with the traditionally vivid colors used in the sixteenth century to represent joyful biblical events. Complementing previous research on the disruption and loss of the red glazes in these paintings, the investigation focused on the analysis of materials, painting technique and degradation issues that further affected the original paintings, changing the viewer's perception and understanding of these artworks. The investigation combined the visual examination of the painting surface with microscopic and spectroscopic analysis of the binder and pigments. A conventional palette made of lead white, lead‐tin yellow, ochres, vermilion, verdigris, smalt, azurite, carbon black and a red lake made of brazilwood and cochineal was identified. The pigments were bound in an oil‐based medium. Chemical and physical alterations detected in paints rich in smalt and verdigris were found to be responsible for color changes affecting significant areas of the compositions. The presence of moisture and the reaction between pigment and binder leading, among other products, to soap and oxalate formation, played a central role in the long‐term behavior of the paint film. Understanding the main degradation processes involved and their consequences is crucial when interpreting an artists' color palette and designing the best approach to preserve these paintings.