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Collecting blood from mice causes artificial hypercorticosteronaemia due to stress.Anaesthesia raises plasma corticosterone levels to up to twenty times above baseline.Tail snip blood sampling causes minimal changes in corticosterone levels.However, repeat tail snip collection causes sustained hypercorticosteronaemia.Unrestrained tail snip sampling should be used for single time point analyses.Accurate measurement of circulating glucocorticoid concentrations in rodents is often hampered by the stress-related activation of the hypothalamic-pituitaryadrenal axis during animal handling. The present study aims to identify methods of blood collection associated with minimal stress and thus artificial increases in plasma glucocorticoid levels. Using two strains of mice, we evaluated common laboratory methods of non-terminal (tail blood sampling with or without restraint; retro-orbital puncture) and terminal blood collection (cardiac puncture) and their immediate and prolonged effect on plasma corticosterone levels.Compared to retro-orbital and cardiac puncture, mice from both the unrestrained and restrained tail snip collection groups displayed the lowest plasma corticosterone levels in both mouse strains. Plasma corticosterone levels in samples obtained from retro-orbital and cardiac puncture collection were up to twenty times higher than those measured in mice undergoing blood collection via tail snip. Repeat tail snip collections (every 30min for 120min, or once after 120min) revealed sustained hypercortisolaemia, compared to the initial collection.We conclude that blood sampling via tail snip without restraint remains the gold-standard method of collection that is associated with minimal stress-related artefacts and hence feasible for single time point corticosterone analyses.