Abstract Biological samples are often frozen and stored for years and/or thawed multiple times, thus assessing their stability on long-term storage and repeated freeze–thaw cycles is crucial. The study aims were to assess:—the long-term stability of two major enzymatic and non-enzymatic metabolites of arachidonic acid, i.e. urinary 11-dehydro-thromboxane-(Tx) B2, 8-iso-prostaglandin (PG)F2α, and creatinine in frozen urine samples;—the effect of multiple freeze–thaw cycles. Seven-hundred and three urine samples measured in previously-published studies, stored at −40 °C, and measured for a second time for 11-dehydro-TxB2 (n = 677) and/or 8-iso-PGF2α (n = 114) and/or creatinine (n = 610) were stable over 10 years and the 2 measurements were highly correlated (all rho = 0.99, P