To evaluate the association between fasting duration and lipid and metabolic test results.A cross-sectional study was conducted in healthy children aged 0-6 years from The Applied Research Group for Kids! (TARGet Kids!) primary care practice network, Toronto, Canada, 2008-2013. The associations between duration of fasting at blood collection and serum lipid tests and metabolic tests were evaluated using linear regression.Among 2713 young children with blood tests the fasting time ranged from 0 to 5 hours (1st and 99th percentiles). Fasting duration was not significantly associated with total cholesterol (β = 0.006; P = .629), high-density lipoprotein (HDL) (β = 0.002; P = .708), low-density lipoprotein (β = 0.0013; P = .240), non-HDL (β = 0.004; P = .744), or triglycerides (β = -0.016; P = .084) adjusted for age, sex, body mass index, maternal ethnicity, and time of blood draw. Glucose, insulin, and homeostasis model assessment of insulin resistance were significantly associated with fasting duration, and the average percent change between 0 and 5 hours was -7.2%, -67.1%, and -69.9%, respectively. The effect of fasting on lipid or metabolic test results did not differ by age or sex; HDL and triglycerides may differ by weight status.In this cohort of healthy young children, we found little evidence to support the need for fasting prior to measurement of lipids. The effect of fasting on glucose was small and may not be clinically important. When measuring serum lipid tests in early childhood, fasting makes a very small difference.ClinicalTrials.gov: NCT0186953.