Fig 1. Experimental design.

13 serum aliquots from each of the 19 male volunteers and a serum pool of all participants were prepared from blood samples; one reference sample from each participant and the serum pool aliquots were immediately frozen in liquid nitrogen; a) 9 aliquots for the experiment of pre-storage handling conditions were kept at RT, on wet ice or on dry ice for 12, 24 or 36 hours and then frozen in liquid nitrogen; b) for the experiment on freeze-thaw cycles three aliquots of each participant were immediately frozen in liquid nitrogen, stored at -80°C and subjected to one to three additional freeze-thaw cycles (FTC: 1 hour thawing at RT, mixing, refreezing at -80°C). All aliquots were subsequently stored at -80°C until analysis by a targeted metabolomics approach.