Background: Metabolites of ethylene glycol (EG) can cross-react in L-lactate assays, leading to falsely elevated serum lactate levels in case of EG intoxication. In this study, we evaluated the effects of EG and its metabolites on routinely used lactate measuring methods. Methods: Serum aliquots were spiked with either L-lactate, EG or one of its metabolites (all 12.5 mmol/L): glyoxal, glycolate, glyoxylic acid or oxalate. An unspiked sample (L-lactate 2.6 mmol/L) served as a control. L-Lactate levels in these samples were measured in 31 national hospitals on 20 different analysers from nine manufacturers. Results: The L-lactate concentrations in the control sample and in the samples spiked with L-lactate, EG, glyoxal and oxalate provided correct results by all routinely used methods. However, the glycolate and glyoxylic acid spiked samples resulted in falsely elevated L-lactate concentration with all blood gas methods and with the majority of general chemistry methods using L-lactate oxidase. Conclusion: The EG metabolites glycolate and glyoxylic acid were shown to falsely elevate L-lactate results with most of the currently used methods due to cross-reactivity with the oxidase enzyme. Falsely elevated L-lactate results can lead to misdiagnosis and inappropriate management of patients with EG intoxication.