During the initial hours of fasting, the body uses up its glycogen stores as an energy source. Once these glycogen reserves are depleted, the body begins breaking down stored triglycerides and structural proteins. During this stage, glycerol becomes a key substrate for gluconeogenesis, while free fatty acids undergo beta-oxidation to provide energy for tissues, such as skeletal muscle. In the fasting state, the body spares protein breakdown as much as possible to conserve muscle and structural proteins. However, as fasting continues, skeletal muscles provide a limited supply of amino acids for gluconeogenesis, which are deaminated in the liver to form glucose. The majority of gluconeogenesis during early fasting relies on glycerol from lipolysis and lactate from anaerobic metabolism.

Glycogen reserves are typically depleted within 12–24 hours, depending on metabolic demands. Once these stores are exhausted, the body shifts to lipolysis in adipose tissue, breaking down stored triglycerides into free fatty acids and glycerol.. This process releases fatty acids for beta-oxidation and glycerol for gluconeogenesis, helping to stabilize blood glucose levels. Simultaneously, muscle fibers and other cells use free fatty acids released by lipolysis to generate energy. The elevated plasma levels of free fatty acids stimulate liver cells to produce ketone bodies, which serve as an alternative fuel source. Ketone bodies, due to their lipid solubility, can cross cell membranes and the blood-brain barrier, providing energy for muscles and neurons.

During prolonged fasting (lasting days to weeks), ketone bodies become the brain's primary energy source, meeting up to two-thirds of its energy requirements. This metabolic adaptation helps conserve muscle protein by reducing the body's reliance on gluconeogenesis for glucose production.