The majority of the sugar in milk is known to be lactose, a disaccharide consisting of a molecule of glucose and one of galactose. In a minority portion of the population, lactose can produce intolerance, due to a deficiency in the enzyme lactase which the human body sometimes stops synthesising after weaning. In most cases the deficiency is due to a prolonged absence of milk and dairy product intake and can be gradually restored. .

There is almost no lactose in medium- and long-matured cheeses as it is the chosen fermentation substrate for the lactic bacteria, during the milk transformation process and also during the ripening of the cheese. Pecorino Toscano PDO has no appreciable lactose content. A recent study showed that the lactose content in fresh pecorino is around 0.4 g/100 g of cheese, whilst it is absent from moulds aged for more than four months, as is also stated on the label.