Graphical AbstractSummary:Our objective was to validate a cow-side meter to estimate blood nonesterified fatty acids (NEFA). Specifically, we evaluated whether NEFA concentrations can be measured in whole blood to omit the step of centrifugation on the farm. Therefore, we measured NEFA concentration in lithium heparinized whole blood and serum and compared results to NEFA measurements using the gold standard method. Results obtained from whole blood were comparable with results measured in serum. Using whole blood and thresholds of 0.3 and 0.4 mEq/L, the meter had a high specificity but a moderate sensitivity. Accuracy was high, however, when using 0.7 mEq/L as the threshold. Furthermore we evaluated the effect of ambient temperature on accuracy of the meter. Compared with measurements at an ambient temperature of 21°C, results were poorer at an ambient temperature of 15 and 6°C.