The potential of the spot urine sampling technique to assess urinary pseudouridine excretion was evaluated. Twelve multiparous Holstein-Friesian cows were fed two experimental diets in a complete change-over design with two 14 day experimental periods. Diets were either silage fed ad libitum with a concentrate supplement offered as a single meal (SF), or a complete diet formulated from the same ingredients (CD). Total urine collections were performed for 24h at 2 h intervals on days 11 and 14. Pseudouridine and creatinine excretion during each 2h interval depended on time of collection (pseudouridine, P