De novo lipogenesis (DNL) is recognised as potentially contributory to pathogenesis of non-alcoholic fatty liver disease (NAFLD), and could be a factor in the progression of simple steatosis to steatohepatitis, although it contributes a relatively minor amount of whole body fat compared to dietary lipid. Consequently understanding the lipidome during induced DNL may provide insight into potential biomarkers of DNL that could be clinically relevant in those at risk of NAFLD and the metabolic syndrome, particularly those of moderate BMI but predisposed to developing fatty liver. Initially to study DNL in vivo, 7 ob/ob mice were fed either high fat diet, to inhibit DNL, or regular chow diet, to stimulate DNL, along with deuterium enriched drinking water. The high resolution liquid chromatography mass spectrometry (LC-MS) and isotope ratio MS demonstrated an overall increase in saturation of fatty acids (FA) in triglycerides, with increased deuterium incorporation into the triglycerides predominantly containing C16:0, C16:1 and C18:1. To translate these results into humans ten healthy men from 21 to 30 years-old were recruited. On the first day participants consumed deuterium-enriched water, labelling lipids produced during DNL. The following day blood samples were drawn before and after a carbohydrate-rich (62% by energy content) meal constituting 110% of calculated basal metabolic rate. Consequently intact lipidomics was performed by LC-MS, and FA composition analysis by GC-MS with and without lipid class separation by solid phase extraction. The rate of de novo lipogenesis was analysed using the incorporation of deuterium into the FA pool of the blood plasma, and this data was integrated with the concomitant changes in intact lipids. In conclusion, these murine experiments demonstrated that intact lipids may be a viable marker of DNL in vivo. Additionally the established deuterium incorporation method for DNL parallels the novel intact lipid markers in the human intervention study.