Advancements in medical care have significantly improved survival rates following major traumatic injury. An understanding of the hormonal, inflammatory and metabolic changes that occur following trauma is still evolving but it is clear that they impact significantly upon patient prognosis. To date, studies that have examined trauma-induced changes in steroid metabolism have analysed samples taken from patients post-hospital admission, culminating in marked variability in the time to first blood sample. Here, via the analysis of pre-hospital blood samples acquired within one hour of injury, we have examined steroid biosynthesis in the immediate aftermath of major trauma. We recruited 30 male trauma patients (mean age 26 years; S.D. ±10 years; range 1959 years) who had an initial blood sample taken within one hour of injury, with subsequent samples taken 412 and 4872 hours post-injury. Blood samples were taken before the administration of analgesics. Morning serum samples obtained from 35 healthy male volunteers (mean 30 years; S.D. ±9; range 1850) served as a control cohort. Steroids were quantified by liquid chromatography-tandem mass spectrometry on a Waters Acquity UPLC chromatography system coupled to a Waters Xevo-XS mass spectrometer and electrospray ionisation source (positive mode). We quantified 18 steroids including glucocorticoid precursors (progesterone, 17-hydroxyprogesterone, 11-deoxycortisol), glucocorticoids (cortisol, cortisone), mineralocorticoid precursors (11-deoxycorticosterone, corticosterone), the major mineralocorticoid aldosterone, classic pathway androgens (dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, testosterone, 5alpha-dihydrotestosterone), and 11-oxygenated androgens (11-hydroxyandrostenedione, 11-ketoandrostenedione, 11-ketotestosterone, 11-hydroxytestosterone). Within minutes of traumatic injury, we observed a steep increase in glucocorticoid and mineralocorticoid outputs while in parallel circulating concentrations of androgens and 11-oxygenated androgens decreased sharply; this steroid profile was sustained at subsequent time points. The relationship of these changes with patient outcome warrants further investigation.