The colour of a paint layer is determined by the pigments mixed with a vehicle and painted on a support. According to their chemical nature, the vehicles are substances that, after drying or hardening, produce an uniform and homogeneous layer. With the aim of quantifying the influence of the medium on the specification of the painting colour, we realized samples of paintings. We considered pigments and dyes of the artist's hues using the vehicles typical of ancient times: oils (especially linseed and poppy oils), casein and egg yolk, mixed according to old recipes. Measurements were performed by reflectance spectrophotometry observing the Spectral Reflectance Factor (%SRF) trend and calculating colour changes. The colorimetric study was carried out in the CIELAB colour space related to the CIE 1964 standard observer and with the D65 illuminant. The results were evaluated also on the basis of the distribution of pigment particles on paint layers observed by scanning electron microscopy.