Background: Repeated freezing and thawing of plasma (or serum) may influence the stability of plasma (or serum) constituents. Despite the alarming warnings from commercial manuals that freeze–thaw cycles affect the stability of hormones in plasma (or serum), surprisingly little, consistent information about this concept is available in literature. Methods: We studied the stability of 15 endocrine parameters (adrenocorticotropic hormone, osteocalcin, plasma renin activity, α-subunits, cortisol binding globulin, glucagon, inhibin B, fT4, TT4, TT3, rT3, TBG, TSH, chromogranin A and thyroglobulin upon repeated freeze–thaw cycles in plasma (or serum) samples from 10 volunteers. Blood was collected by venipuncture and after centrifugation and aliquoting, all samples were frozen at −20℃. Aliquots were thawed up to four times and changes in concentrations of endocrine parameters were compared to baseline condition. Results: Repeated freeze–thaw cycling resulted in significant and relevant increases of plasma renin activity and a small decrease of adrenocorticotropic hormone. Conclusions: For most of the analysed endocrine parameters, we found no effects of multiple freeze–thaw cycles despite alarming notifications in assay manuals. Plasma renin activity was the only endocrine parameter that showed significant and relevant changes following repeated freeze–thaw cycling.