Background The lack of association between serum testosterone levels and symptoms suggestive of hypogonadism is a significant barrier in the determination of late-onset hypogonadism (LOH) in men. This study explored whether testosterone levels increase after morning awakening, likewise the cortisol awakening response (CAR) in the hypothalamic–pituitary–adrenal (HPA) axis, and whether testosterone levels during the post-awakening period are associated with age and symptoms suggestive of late-onset hypogonadism (LOH) in men. Methods Testosterone and cortisol levels were determined in saliva samples collected immediately upon awakening and 30 and 60 min after awakening, and scores of the Aging Males’ Symptoms (AMS) questionnaire were obtained from 225 healthy adult men. Results A typical CAR (an increase in cortisol level ≥ 2.5 nmol/L above individual baseline) was observed in 155 participants (the subgroup exhibiting typical CAR). In the subgroup exhibiting CAR, testosterone levels sharply increased during the post-awakening period, showing a significant negative correlation with age, total AMS score, and the scores of 11 items on the somatic, psychological, and sexual AMS subscales. Of these items, three sexual items (AMS items #15–17) were correlated with age. Meanwhile, there was no notable increase in testosterone levels and no significant correlation of testosterone levels with age and AMS score in the subgroup exhibiting no typical CAR (n = 70). Conclusions The results indicate that the hypothalamus-pituitary-gonad (HPG) axis responds to morning awakening, and determining testosterone levels during the post-awakening period in men with typical CAR may be useful for assessing HPG axis function and LOH.