Purpose: This study investigated the influence of manual acupuncture on heart rate variability and the role which anxiety can play in modifying physiological outcomes. Method: Analysis of heart rate variability (HRV) was used as a sensitive and a reliable indicator of the balance between sympathetic and parasympathetic regulation of the heartbeat. Two groups of healthy female subjects were recruited into the study. The control group (n = 30) attended one experimental session where no acupuncture treatment was used. The experimental group (n = 30) attended three sessions in which unilateral manual stimulation of acupuncture points LU7 and KD6 was performed. Results: The stimulation of the acupuncture points LU7 and KD6 was not associated with significant changes in HRV. Previous familiarity with acupuncture did not influence the outcomes but level of anxiety had a strong impact on physiological outcomes. Stimulation of LU7 acupuncture point counterbalanced naturally occurring sympathetic increase over time and had relaxing and harmonizing effect on the heart rhythm without influencing subjective perception of increased anxiety. Stimulation of KD6 acupuncture point had sympathetic influence on HRV in subjects with low "trait" anxiety and this influence was nullified by simultaneous stimulation of LU7 acupuncture point. Conclusions: It seems likely that the level of anxiety can modify HRV during acupuncture treatment and up to 40 min after the treatment. Psychological factors such as anxiety level should be considered as having important influence on physiological response to acupuncture.