© 2019 S. Karger AG, Basel. All rights reserved.Background: Connective tissue manipulation (CTM) has therapeutic effects on diseases with autonomic imbalance, yet its mechanisms of action are not clearly identified. Objective: The aim of this study was to investigate acute autonomic responses to CTM in healthy young women with various physical activity levels. Method: The study was designed as a nonrandomized, controlled single-center study. Healthy women aged between 18 and 25 years were assigned to a connective tissue manipulation group (CTMG) (n = 150) or a control group (CG) (n = 60). CTM was applied to the CTMG while the CG did not receive any intervention. Respiratory rate (RR), heart rate, systolic/diastolic blood pressures (SP/DP), oxygen saturation (OS) and body temperature were measured. The International Physical Activity Questionnaire short form was used to determine physical activity levels. -Results: Analysis revealed significant reduction in SP and DP and an increment in RR in the CTMG (p < 0.05). RR increased and SP decreased among inactive, SP decreased among moderately active, and SP and OS decreased among highly active participants in the CTMG (p < 0.05). There were no significant alterations in the CG (p > 0.05). Conclusions: CTM has an immediate reducing effect on sympathetic activity in healthy young women independently from the physical activity level. Future studies are needed to clarify long-term effects of CTM on autonomic functions in healthy individuals.