Deuterated water (2H2O) can be used to measure de novo lipogenesis (DNL) with minimal participant burden, but implications of tracer washout on repeated measures in humans are unclear. This study is an exploratory analysis of data from a crossover trial to determine the impact of duration between repeated 2H2O dosing for sequential assessments of DNL, alongside day-to-day variability in DNL. Twenty-two non-obese adults (11 men, 11 women) completed three laboratory visits in a randomised, crossover design (35±13-day washout). participants consumed 3 g·kg body water-1 of 2H2O the evening before laboratory visits. Blood was sampled before 2H2O ingestion, and the following morning in a fasted state. 2H enrichment of plasma water and very-low density lipoprotein-triacylglycerol (VLDL-TG)-palmitate were used to determine DNL. Whilst pre-dosing plasma water 2H enrichments (mole percent) increased across visits from 0.017 ± 0.003% to 0.022 ± 0.008% and 0.027 ± 0.014%, respectively (p<0.05), post-dose enrichments did not display a systematic bias, and nor did 2H enrichments of VLDL-TG-palmitate or measures of DNL (largest mean difference: -1.2%; 95%CL:-12.7 to 9.1%, p=0.45). The day-to-day SD and CV% of fractional hepatic DNL was 2.39% (95%CI:1.35 to 3.42%) and 27% (95%CI:19 to 35%), respectively. Repeated 2H2O dosing does not systematically bias measures of fasting hepatic DNL when using a washout duration of ~4 weeks. We also found no evidence that DNL is biased by washout durations of 3 weeks. Therefore, 2H2O can be used to reliably assess human hepatic DNL in repeated measures designs with at least three weeks between sequential measures.