To evaluate diurnal variation of plasma bone markers, blood samples were collected from five calves at 2-hr intervals throughout a 24-hr period. Tartrate-resistant acid phosphatase isoform 5b (TRAP5b), carboxy-terminal collagen crosslinks of type-I collagen (CTX), hydroxyproline, bone specific alkaline phosphatase (BALP) and osteocalcin were measured. Cosinor analysis showed a significant rhythm in all bone markers. The acrophase of each bone marker appeared from the early to late morning. The percentage ratio of the amplitude to mesor and the within-subject variability for CTx and osteocalcin were significantly larger than those for TRAP5b and BALP. This marked diurnal variation in five bone markers suggested that the time of blood sampling should be fixed when studying bone marker concentrations in bovine plasma.