This study aimed to determine the effect of yoga stretching on salivary stress hormones and cardiac autonomic nervous system. To our knowledge, this study is the first to investigate changes in cardiac autonomic nervous system after yoga stretching. In this crossover design study, 10 adult men (age, 26.3 ± 2.5 years) without yoga experience participated in the rest and yoga trials for 90 min. Measurements were carried out before (pre), immediately (post), 60 min, and 120 min after rest or yoga stretching. Saliva samples were collected by chewing a sterile cotton ball at a frequency of 60 cycles per minute. Salivary cortisol and testosterone concentrations were measured using an enzyme-linked immunosorbent assay. With the subjects in the sitting position, heart rate variability was measured using pulse analyzer plus for 150 seconds. As regards rate changes, salivary testosterone level tended to increase (p = 0.088), testosterone/cortisol ratio significantly increased (p < 0.05), and cortisol level significantly decreased (p < 0.05) at 120 min after yoga stretching. The square root of the mean-squared differences of successive normal-to-normal intervals and natural logarithm high-frequency component, which are indicators of parasympathetic nerve activity, increased at 60 min (p < 0.05) and 120 min (p < 0.05) in the yoga trial, respectively. In conclusion, yoga stretching can enhance parasympathetic nerve activity and improve stress hormones. Therefore, yoga stretching may be useful to compensate for physical inactivity and increase life expectancy in the general population.