During the neonatal period, high protein breakdown rate is a metabolic process inherent to elevated rates of protein accretion in skeletal muscle. To determine the relationship between hindlimb net movements of essential and non-essential amino acids in the regulation of hindlimb protein breakdown during overnight fast and fed cycle, overnight food-deprived 10- and 28-d-old piglets were infused (7 h) with [1-^13C]phenylalanine and [ring-²H₄]tyrosine during a 3-h period and then during a 4-h feeding period. Extraction rates for aspartate and glutamate after an overnight fast were 15% and 51% in the 10-d-old compared with 6% and 25% in the 28-d-old (P < 0.05), suggesting an altered requirement for precursors of amino acids shuttling nitrogen to the liver as early life progresses. This was simultaneous to marginal positive hindlimb net balance of essential amino acids after an overnight fast, with negative net release of many non-essential amino acids such as alanine, asparagine, glutamine, glycine, and proline. This suggests that newborn muscle does not undergo significant protein mobilisation after short period of fasting in the support of elevated protein accretion rate. Furthermore, tyrosine efflux from hindlimb breakdown between overnight fast and feeding periods were not different in the 10-d-old pigs for whom tyrosine was limiting, but when tyrosine supply balanced requirements in the 28-d-old piglet, the hindlimb efflux was increased (P = 0.01). The study indicates that proteolysis and net movements of amino acids are coordinated mechanisms that sustain the elevated rate of net protein accretion during overnight feeding-fasting cycles in the neonate.