Introduction: Several eicosanoids and other oxylipins are important lipid mediators. Reliable quantification in plasma is important to assess the state of disease, action of drugs and the biology of oxylipins. In order to monitor biological changes, low background variability of oxylipin concentrations in biological samples is essential for proper interpretation of oxylipin biology. However, only little is known about the variation in the oxylipin profile in healthy human subjects. Experimental: Inter-day variation in circulating oxylipins after overnight fasting was investigated in healthy young men on either a standardized or non-standardized diet during a (24 to) 48 h time interval. Intra-day variance was investigated during an 8 h time interval (covering breakfast and lunch meals) in men on a standardized diet with blood sampling at 0, 2, 4, 6 and 8 hours. Free oxylipins in plasma were analyzed using a targeted metabolomics platform for the quantification of 160 oxylipins from different precursors. Analytical variation was evaluated based on quality control plasma samples. Results: Free oxylipins in quality control plasma samples showed low variations (<20% for most analytes). Inter-day variations in fasting blood were in the same range, while significant differences were observed within the day (intra-day variance). Conclusion: Based on the low intra-individual inter-day variance in concentrations of free oxylipins, our results demonstrate the suitability of fasting plasma for the investigation of oxylipin biology. In non-fasting plasma samples, the variations were high during the day. Thus, non-fasting plasma samples appear to be unsuitable to evaluate biologically relevant changes, for instance, those caused by disease or drugs. However, it remains to be determined if the same standardized meal results in reproducible modulations of the oxylipin profile allowing evaluation of the oxylipin pattern during the postprandial state.