Objective: This study aims to compare the instantaneous effects of chiropractic upper and middle thoracic manipulations on the autonomic nervous system. Material-Method: The study included 30 healthy volunteers aged 18-45 years. Manipulation was applied to the upper thoracic (T1-4) area (n=15, 8 female, 7 male) and to the middle thoracic (T6-9) area (n=15, 7 female, 8 male). Before the application, heart rate, body temperature and skin conductivity (EDA) measurements were performed using the Empatica E4 wristband device. Thoracic HVLA (high velocity low amplitude) manipulation was performed in the supine position. After the application, the measurement made with the Empatica E4 device was repeated. The mean body temperature and skin conductivity values were calculated for the analysis of the data. Heart rate data were evaluated using Kubios heart rate variability (HRV) standard program. Detailed information about the effects of upper and middle thoracic area manipulation on the autonomic nervous system was obtained according to the analysis results of the data obtained before and after manipulation. Correlation analysis of pre- and post-manipulation data and difference analysis between the two groups were performed based on significance level p<0.05. Results: As a result of the research, no significant changes in parasympathetic nervous system values such as RMSSD, pNN50, PNS Index and sympathetic nervous system values such as Stress Index and SNS Index were observed after upper thoracic manipulation. After middle thoracic manipulation, significant decreases occurred in sympathetic nervous system values such as Stress Index, SNS Index, while significant increases occurred in parasympathetic nervous system values such as RMSSD, pNN50, PNS Index. It was observed that there was no significant change in temperature and EDA. Conclusion: As a result of our measurements and analyzes, it was seen that chiropractic upper and middle thoracic manipulations had different instantaneous effects on the autonomic nervous system.