To investigate processing- and storage-dependent changes in D- and L-lactate concentration, blood samples from eleven healthy Holstein calves were spiked with 3 mM D-lactic acid and 3 mM L-lactic acid immediately following collection (time 0) or left untreated for comparison. Serum and plasma, respectively, were separated 0.5 hours following collection or left in contact with blood cells, stored at 4°C and analyzed for D- and L-lactate concentration using enzymatic assays at 1, 2, 4, 8, 12, 24, and 48 hours. Concentrations were compared to the 1 hour sample. D- and L-Lactate concentrations in all separated samples were stable for up to 48 hours. When left in contact with cells, L-lactate concentration in untreated and spiked serum and in spiked plasma, D-Lactate concentration in untreated serum, and total lactate concentration in untreated serum increased significantly by 48 hours.