© 2019, Springer Science+Business Media, LLC, part of Springer Nature. Background: In our metabolomics studies we have noticed that repeated NMR acquisition on the same sample can result in altered metabolite signal intensities. Aims: To investigate the reproducibility of repeated NMR acquisition on selected metabolites in serum and plasma from two large human metabolomics studies. Methods: Two peak regions for each metabolite were integrated and changes occurring after reacquisition were correlated. Results: Integral changes were generally small, but serum citrate signals decreased significantly in some samples. Conclusions: Several metabolite integrals were not reproducible in some of the repeated spectra. Following established protocols, randomising analysis order and biomarker validation are important.