BACKGROUND: Ultrasound (US)-guided percutaneous needle electrolysis (PNE) is a novel minimally invasive approach, which involves the application of a galvanic current via an acupuncture needle. As in any procedure involving needling, vagal reactions have been reported during PNE. OBJECTIVE: To examine for changes in autonomic activity during the US-guided PNE technique on healthy patellar tendons by measurement and analysis of heart rate variability (HRV). METHODS: Twenty-two male footballers were randomly allocated to: a control group (11 players), for whom HRV was recorded for 10 min, both at rest and during an exhaustive US examination of the patellar tendon and adjacent structures; and an experimental group (11 players), for whom HRV was recorded for 10 min, both at rest and during application of US-guided PNE on the patellar tendon. The following HRV parameters were assessed: mean NN interval, mean heart rate, time domain parameters (SDNN, rMSSD, pNN50), diameters of the Poincare plot (SD1, SD2), stress score, and sympathetic/parasympathetic ratio. RESULTS: There were no differences between groups in any baseline measurements, nor were there any significant differences between control group measurements (baseline vs intervention). The experimental group exhibited statistically significant increases in SDNN/SD1 (p=0.02/p=0.03) and SD2 (p=0.03), indicating increased parasympathetic and decreased sympathetic activity, respectively. CONCLUSIONS: US-guided PNE was associated with an autonomic imbalance characterised by greater parasympathetic activity, which could potentially result in a vasovagal reaction. Care should be taken to monitor for adverse reactions during US-guided PNE and simple HRV indicators may have a role in early detection.