Background Blood profile testing is commonly used to monitor herd health status, diagnose disorders, and predict the risk of diseases in cows and calves, with subsequent optimization the production of dairy herds. By understanding the physiological ranges of serum metabolites relative to age, lactation stage, and the sampling time in healthy cows and calves, the dairy practitioners can accurately diagnose abnormalities with a blood test. The effect of sampling time on the variation of serum metabolites within 24 h were evaluated in 83 cattle. All animals were originated from a dairy herd, where the animals, based on their ages and lactation stages, were classified into eight groups. The blood samples were collected from each animal every 4 h within a day. Results The time of sampling within the day showed significant influences on the serum concentrations of glucose, beta-hydroxybutyric acid (BHBA) and urea. BHBA was the most metabolite that showed day variation among cows' groups. Furthermore, the concentrations of total cholesterol were the most stable metabolite in all groups. The mean values of albumin, total proteins, glucose, non-esterified fatty acids (NEFA), BHBA, total cholesterol, total bilirubin, urea, and creatinine revealed significant variations among the different studied groups. Conclusions A certain suitable time of blood sample collection cannot be recommended. However, care shall be taken for the time of sampling for measurements of glucose, NEFA, BHBA and urea, otherwise the comparative values of these metabolites at different sampling time points may differ significantly from each other's, without a disease cause. It may be recommended, for metabolic assessment of dairy herds, classification the subjects into different groups based on lactation stages and ages of animals.