Steroid biosynthesis and biotransformation are based on a cascade of enzymatic processes being highly sensitive to various external influences. Amongst those, ethanol was shown to affect testosterone metabolism. For doping analyses, athlete steroid profiles comprise seven urinary steroid metabolites, of which relevant ratios are significantly increased following ethanol consumption. This effect is presumably based on the lack of hepatic NAD+-coenzyme as a consequence of ethanol oxidation. Only recently, testosterone (T) and androstenedione (A4) blood profiles have been introduced as additional approach for doping control. However, a potential influence of ethanol intake on testosterone biosynthesis and thus on blood steroid profiles has not been investigated so far. Therefore, steroid concentrations from 10 males and 10 females receiving an ethanol infusion up to a breath alcohol concentration of 0.5mg/L which was hold as a plateau for two hours were conducted. Blood samples were drawn every 15minutes for steroid quantification. An ethanol-dependent T/A4 increase up to 385% resulting from A4 suppression was observed in 14 volunteers. In addition, we observed sporadic A4 increases coinciding with cortisol and ACTH pulses pointing to a meal-induced adrenal stimulation. While testosterone levels in males showed diurnal variation solely, testosterone levels in some females were found to be susceptible to ethanol- and ACTH-dependent perturbations, which is thought to be due to its predominant adrenal synthesis in females. In conclusion, the results of the present study emphasize the importance of blood sampling at a sufficient time interval from food and ethanol intake. This is of interest if T and A4 are used for diagnostics in doping control.