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Two trials were conducted to examine factors potentially influencing the measurement of blood β-hydroxybutyric acid (BHBA) in dairy cows. The objective of the first trial was to study effects of sampling time on BHBA concentration in continuously fed dairy cows. Furthermore, we determined test characteristics of a single BHBA measurement at a random time of the day to diagnose subclinical ketosis considering commonly used cut-points (1.2 and 1.4 mmol/L). Finally, we set out to evaluate if test characteristics could be enhanced by repeating measurements after different time intervals. During 4 herd visits, a total of 128 cows (8 to 28 d in milk) fed 10 times daily were screened at 0900 h and preselected by BHBA concentration. Blood samples were drawn from the tail vessels and BHBA concentrations were measured using an electronic BHBA meter (Precision Xceed, Abbott Diabetes Care Ltd., Witney, UK). Cows with BHBA concentrations ≥0.8 mmol/L at this time were enrolled in the trial (n=92). Subsequent BHBA measurements took place every 3h for a total of 8 measurements during 24 h. The effect of sampling time on BHBA concentrations was tested in a repeated-measures ANOVA repeating sampling time. Sampling time did not affect BHBA concentrations in continuously fed dairy cows. Defining the average daily BHBA concentration calculated from the 8 measurements as the gold standard, a single measurement at a random time of the day to diagnose subclinical ketosis had a sensitivity of 0.90 or 0.89 at the 2 BHBA cut-points (1.2 and 1.4 mmol/L). Specificity was 0.88 or 0.90 using the same cut-points. Repeating measurements after different time intervals improved test characteristics only slightly. In the second experiment, we compared BHBA concentrations of samples drawn from 3 different blood sampling locations (tail vessels, jugular vein, and mammary vein) of 116 lactating dairy cows. Concentrations of BHBA differed in samples from the 3 sampling locations. Mean BHBA concentration was 0.3 mmol/L lower when measured in the mammary vein compared with the jugular vein and 0.4 mmol/L lower in the mammary vein compared with the tail vessels. We conclude that to measure BHBA, blood samples of continuously fed dairy cows can be drawn at any time of the day. A single measurement provides very good test characteristics for on-farm conditions. Blood samples for BHBA measurement should be drawn from the jugular vein or tail vessels; the mammary vein should not be used for this purpose.