OBJECTIVES: This study aims to comprehensively investigate the changes of salivary stress biomarkers, psychological status, and autonomic nervous system (ANS) response due to horticultural activities (HAs). DESIGN AND METHODS: A prospective observational study was conducted in twenty Japanese healthy adults (mean age, 58.4 years). Flower appreciation, flower arrangement, and farm work experience were performed as three HAs with different working concepts. Five salivary stress biomarkers (cortisol, α-amylase, S-IgA, chromogranin A, and oxytocin) were measured to quantify the stress levels before and after each HA. The Profile of Mood Status 2nd edition (POMS2) was used as a subjective psychological evaluation. Wearable biosensors were used to visualize the continuous ANS response throughout the process. RESULTS: In the POMS2 investigation, the negative factors, which included Anger-Hostility, Confusion-Bewilderment, Depression-Dejection, Tension-Anxiety, and Total Mood Disturbance, were significantly decreased (p=0.0135, p=0.0004, p=0.0024, p=0.0015, p=0.0063, respectively). In the measurement of salivary stress biomarkers, flower appreciation decreased cortisol (p=0.0134), and farm work experience not only decreased cortisol but also increased oxytocin (p=0.0041, p=0.0128 respectively). In the visualization results of the ANS response, a graph demonstrated that the difference in activity between the sympathetic nerve and the parasympathetic nerve was narrowed by a series of HAs. CONCLUSIONS: In healthy adults, HAs had a stress-reducing effect, which was evidenced by neuroendocrinological and psychological evaluations, a study of POMS2, salivary stress biomarkers, and visualization of the ANS response.