Giotto used cow milk to prepare walls to be frescoed.
The discovery was made after examining fragments of frescoes by Giotto and his master Cimabue which collapsed when Assisi's Basilica of St Francis was hit by a 1997 earthquake.
A Naples University biochemical team led by Pietro Pucci analysed the fragments and found proteins from cow's milk.
''We developed non-invasive techniques to study the composition of the substances used to prepare the base of the frescoes and stick on the super-thin gold leaf used for the haloes,'' Pucci told a conference here Wednesday.
''In this way we were able to isolate the milk proteins,'' said Pucci, whose study with Perugia University colleagues Antonio Sgamellotti and Laura Contechini will be published shortly.