This cross-sectional study compared hormonal responses to resistance exercise between trained and untrained men to investigate the adaptations of the endocrine system to long-term strength training in middle-aged men. Twenty-one middle-aged men were recruited for this study and matched into a strength-trained group (SG) (n = 10) and an untrained group (UG) (n = 11). In the SG, the individuals had practiced strength training for hypertrophy for at least 3 years. Upper- and lower-body muscle strength was measured with a 1 repetition maximum (1RM) test. Blood samples were collected at rest and after multiple sets of a superset strength training protocol (SSTP), with an intensity of 75% of 1RM values. With these blood samples, the levels of total testosterone (TT), free testosterone (FT), dehydroepiandrosterone (DHEA), cortisol, and sex hormone-binding globulin (SHBG) were determined. In addition, the TT-to-cortisol ratio and TT-to-SHBG ratio were calculated. There was no difference at rest between groups in hormonal values for TT, FT, DHEA, cortisol, the TT-to-SHBG ratio, and the TT-to-cortisol ratio. There were increases after SSTP in the levels of TT, FT, DHEA, and cortisol and the TT-to-SHBG ratio in the UG, but only FT increased in the SG. The SG demonstrated lower values in the TT-to-SHBG ratio after the training session. These results suggest the presence of alterations in anabolic and catabolic hormonal responses to resistance exercise in long-term trained middle-aged men, with the trained subjects demonstrating lower responsiveness in the hormone values. Long-term trained men seem to require a higher volume of training, at least similar to their daily workout, to stimulate greater hormone responses.