BACKGROUND: Heart rate variability (HRV) has been shown to be influenced by several factors such as noise, sleep status, light, and emotional arousal; however, little evidence is available concerning autonomic responses to a venipuncture. The purpose of this study was to investigate changes of HRV indexes and heart rate (HR) during and following a venipuncture procedure among healthy individuals. METHODS: 33 healthy individuals (22.8 +/- 0.56 years, 167 +/- 1.56 cm, 69.5 +/- 2.61 kg) participated. Testing included 10-minute HRV analysis prior to the venipuncture, a 1-minute venipuncture procedure followed by a 10-minute analysis of HRV, and a total recording of 21 minutes. The first 5 minutes of the 21-minute recordings were discarded, and the remaining 5 minutes of the resting segment was analyzed (PRE), and the last 5 minutes of the 21-minute recording (POST). The log transformation of the time domain root mean squared of successive differences (lnRMSSD) and the frequency domains of high frequency (lnHF) and low frequency (lnLF) and LF/HF ratio (lnLF/HF) were used to quantify autonomic activity. HR was measured in 1-minute segments at 2 minutes prior (PRE), venipuncture (STICK), and post (P1-5). RESULTS: HR significantly increased at STICK (P = 0.002), and fell below resting at P-5 (P < 0.001). lnRMSSD and lnHF increased significantly by POST (P < 0.001, P = 0.005). lnLF/HF ratio significantly decreased at POST (P = 0.047), while no significant changes occurred for lnLF (P = 0.590). CONCLUSIONS: HRV and HR are influenced for 10 minutes following the venipuncture procedure. Practitioners and researchers who are interested in collecting blood and measuring HRV need to account for the influence of the venipuncture.