Background: Middle-aged women often suffer from mood symptoms such as depression and anxiety, which may be associated with autonomic modulation. Stretching exercises improve joint flexibility, mood symptoms, and parasympathetic activity. However, the effects of stretching on mood symptoms and autonomic modulation in middle-aged women are unknown. Thus, this study investigated this.Methods: Altogether, 25 middle-aged women (51.8 +/- 4.4 years) were included in this study. Each participant completed a whole-body stretching program for 25 min or a control resting period in random order on separate days. Mood symptoms, including negative or positive mood and anxiety, were assessed before and after the intervention using the Profile of Mood States-2 and the State-Trait Anxiety Inventory. Autonomic modulation was measured using heart rate variability before, immediately after, and 30 min after the intervention. A two-way repeated-measures analysis of variance was performed for comparisons. Post hoc testing was performed using the Bonferroni correction.Results: Mood symptoms significantly improved after stretching, especially in terms of decreasing anxiety and inducing a positive mood (p = 0.001 - 0.047). However, the autonomic modulation indices showed no significant changes immediately or 30 min after stretching under either the stretching or control conditions (p = 0.068 - 0.808).Conclusion: Stretching exercises improved mood symptoms, particularly anxiety and positive moods. However, the autonomic modulation was not altered. Stretching is an effective exercise for managing mood and mental health in middle-aged women.