As grain prices rise, the search for alternative glycogenic precursors in animal feed becomes increasingly important, and this study was conducted to determine if the replacement of starch with glycerol, as an alternative glycogenic precursor, affects the milk metabolic profile and milk coagulation ability, and therefore the quality of the milk. Eight primiparous mid-lactation Holstein cows were fed during a replicated 4 × 4 Latin square trial with four different isoenergetic rations: (1) control (T0) fed a total mixed ration (TMR) with barley meal; (2) group T1, decreased barley content, replaced isoenergetically with 1 kg crude glycerol; (3) group T2, the barley meal was replaced with 2 kg of crude glycerol; and (4) group T3 the barley meal was replaced with 3 kg of crude glycerol. Rumen, blood and milk samples were collected at the end of every 21-d treatment period. Rumen samples were analysed for proportion of total volatile fatty acid (VFA), blood samples for insulin and glucose, and milk for metabolites (e.g. citric-acid cycle compounds). The change in glycogenic precursors had a positive effect on rumen VFA proportions; the proportion of propionic acid increased (P < 0.001). Milk protein (P < 0.001) and curd firmness (P < 0.001) both increased. The increase in milk protein concentration may have been due to an increase in microbial protein. Regarding the milk metabolic profiles, different signals were positively associated with coagulation ability and change in the diet. Based on this study, changing the glycogenic precursor in animal diet in this way is possible, and may have no immediate deleterious consequences on milk quality or cow health. Indeed, there is evidence for benefits from this substitution.