Currently, subclinical metabolic imbalances at the individual cow and herd level are detected by measuring biomarkers in single blood samples. However, diurnal variations have not been fully described yet but need to be considered when sampling for a robust ad consistent analysis. The study describes the influence of lactation phases on circadian rhythms and diurnal variations for non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), total bilirubin (tBIL) and aspartate aminotransferase (AST) in dairy cows. In an observational pilot study, we used 16 clinically healthy Simmental dairy cows subdivided in four different lactation stages (dry-off, fresh, high and late lactating). Every cow was monitored for 24 h, with blood sampling and assessment of clinical parameters every 2 h. Time and lactation stage influence the concentration of the biomarkers NEFA, BHB and tBIL in serum. Further, circadian rhythmicity was found in high lactating cows for NEFA peaking at 5:39 am and BHB peaking at 4:20 pm. We suggest blood sampling for single-point measurements within three hours after the first feeding until two hours after the last feeding of the day. The results provide a new insight into the physiology of circadian rhythms in dairy cows and enable improved metabolic monitoring.