Testing of the adrenal function with ACTH 1–24 (Synacthen test) or insulin (insulin tolerance test-ITT) is commonly used. The question of ongoing debate is the dose of Synacthen. Moreover, it may be important from the physiological point of view besides measurement of cortisol levels and 17α-hydroxy-progesterone to know also the response of other steroids to these test. The plasma levels of 24 free steroids and their polar conjugates were followed after stimulation of 1 μg, 10 μg and 250 μg of ACTH 1–24 and after insulin administration in thirteen healthy subjects. The study aimed to describe a response of steroid metabolome to various doses of ACTH 1–24 and to find the equivalency of these tests. The additional ambition was to contribute to understanding of physiology of these stimulation tests and suggest an additional marker for HPA axis evaluation. No increase of most conjugated steroids and even decrease of some of them during all of the Synacthen tests and ITT at 60th min were observed. The levels of steroid conjugates decreased in ITT but did not during all of the Synacthen tests by 20 min of each test. Testosterone and estradiol did not increase during the Synacthen tests or ITT as expected. The results suggest that the conjugated steroids in the circulation can serve as reserve stock for rapid conversion into free steroids in the first minutes of the stress situation. Various doses of ACTH 1–24 used in the Synacthen tests implicate earlier or later occurrence of maximal response of stimulated steroids. The equivalent dose to ITT and standard 250 μg of ACTH 1–24 seemed to be dose of 10 μg ACTH 1–24 producing the similar response in all of the steroids in the 60th min of the test.