The purpose of this study was to describe autonomic nervous system responses to serial connective tissue massage (CTM) in healthy middle-aged and elderly subjects. Fourteen healthy middle-aged or elderly subjects (means age = 61.3 years) were randomly assigned to either a CTM or placebo group. Subjects received nine separate treatments (CTM or placebo) on alternate days over a three-week period. Each treatment was divided into 15-minute control, intervention, and recovery periods. Subjects in the CTM Group (n = 8) were given CTM on the basic section (low back), and subjects in the Placebo Group (n = 6) had sham ultrasound applied to the same area. Variables monitored were skin temperature (right great toe and popliteal fossa), galvanic skin response, mean arterial blood pressure, and heart rate. Variable measurements were made every five minutes. An analysis of variance and single-case analyses showed no changes in the variables measured in the CTM or Placebo Group. The results of this study suggest that CTM has no consistent immediate or long-term effects on the autonomic nervous system in healthy middle-aged and elderly subjects.