Cardiovascular disease is one of the most important causes of death and disability in the world. An autonomic imbalance is associated to cardiovascular risks and disorders. Electrical stimulation (ES) applied by surface electrodes is a non-invasive therapeutic approach with low side-effects. In the last years, some studies had investigated the effects of ES on the autonomic nervous system and cardiovascular system. However, different intensities, frequencies and electrode locations had been used, which makes difficult to clarify the optimal parameters.

The aim of this study is to analyze the effects of ES on autonomic nervous modulation in healthy subjects comparing three different locations of application for ES.

Participants will be aleatory assigned to one of three groups for a single-session of 20-minute transcutaneous ES: a) an auricular intermittent ES in an area that is believed to be connected to the vagal nerve (cymba conchae); b) an auricular sham intermittent ES, meaning a real ES but applied on an ear location not connected to the vagal nerve (scapha); and c) an upper back continuous ES (C7-T4), a placement that is thought to be connected to the stellate (sympathetic) ganglion. All the protocols will be stated at a non-painful intensity. After that, the hand grip exercise will be executed.

The heart rate variability (HRV) and heart frequency (HR) will be measured in four main times: the baseline, in the final minutes of the ES, after the ES and after finishing the hand grip exercise. Blood pressure will be measured also at all the time points except during the ES due to the possibility of interferences. Adverse effects will be assessed after ES and 48 h later. The study will provide initial knowledge about how different electrical stimulation locations contribute to reduce sympathetic excitation and improve the sympathovagal balance.

Electrical stimulation (ES) of the nervous system, also denominated as neuromodulation, has been investigated for diverse conditions as cardiovascular diseases, chronic pain and psychiatric conditions. It is unknown which locations and parameters can be more effective and better tolerated. The transcutaneous ES is a non-invasive technique which has fewer side-effects than subcutaneous vagus nerve stimulation.

The aim of this study is to analyze the acute effects of transcutaneous ES on autonomic nervous modulation by heart rate variability (HRV), heart frequency (HF) and blood pressure (BP) in healthy subjects comparing three different locations for the ES, before and after a hand-grip exercise. In addition, the tolerance to the current, the side-effects and the perceived exertion will be compared.

Once the participants have given their informed consent and had been checked for exclusion criteria, are invited to an experimental session. Subjects will be advised to refrain from caffeine or alcohol for 12 h and vigorous exercise for at least 24h prior to the intervention.

The participants will remain sitting during the entire session and will be instructed to stay awake and avoid movement. Grip strength will be evaluated by a hydraulic dynamometer (Saehan model SH5001). Participants will remain seated on a chair with their feet and trunk supported, shoulders adducted, elbows flexed at 90º, forearms in neutral position, and wrists with 0º-30º extension. Subjects will be instructed to perform a maximum isometric contraction for three to five seconds, and the peak force will be recorded. Three evaluations will be made with an interval of one minute between them. If the examiner would recognize some compensatory movement by the participant, a new measurement would be taken and registered.

The volunteers will be randomized by sex to one of three groups: cymba conchae at the right ear (active group); C7-T4 placement (active group), scapha at the right ear (sham-group). These three arms will be denominated as Transcutaneous Vagus Electrical Nerve Stimulation (t-VNS); Transcutaneous Sympathetic Ganglion Electrical Nerve Stimulation (t-SNS); and Sham Transcutaneous Vagus Electrical Nerve Stimulation (sham t-VNS), and. Allocation concealment, stratified by sex, will be ensured by the use of sealed envelopes containing the treatment allocation. The sequence will be generated using a computer program by a researcher not implicated in the experimental sessions. The envelopes will be opened just before determining the amplitude for stimulation. The stimulation location will be wiped down with alcohol and the minimum intensity, at which the stimulus is perceived, will be evaluated for the auricular locations, whereas minimum amplitude for muscle contraction between the electrodes will be assessed in cervico-thoracic location.

After 15 minutes of resting, the first assessment (baseline) will be taken (HRV, HR and blood pressure) for 5 minutes. The respiratory frequency will be checked every 5 minutes.

Then the randomized ES will be applied for 20 minutes using the same TENSMED S82 (Enraf Nonius, The Netherlands) symmetrical and rectangular biphasic pulses, at 350 microseconds of phase duration. It will be applied at 10 Hz (t-VNS and sham t-VNS) or 5 Hz (t-SNS). Both the two protocols applied to the auricular region (t-VNS and sham t-VNS) will be delivered by trains, whereas a continuous ES will be selected for the (t-SNS). The minimum amplitude for auricular stimulations (t-VNS and sham t-VNS) will be twice the perception threshold, at a comfortable intensity under the discomfort threshold (pain onset). It will be increased during the stimulation only if the sensation is clearly decreased to a maximum of 9 mA. The perception threshold will be set increasing amplitude by 0.5 mA each 3 seconds from 0 mA. Amplitude in t-SNS will be set at a muscle contraction at a comfortable intensity (under discomfort threshold or pain onset). The motor threshold will be determined by increasing amplitude 0.5 mA each 3 seconds from the perception threshold until a motor contraction is observed. The initial amplitude will be 2 mA over this threshold. It will be increased during the stimulation only if the stimulus falls down (to a maximum of 25 mA).

Another 5-minute period after ES will be recorded and then the 3-minute hand grip exercise (SH5001, Saehan, Korea) at 30% of maximum strength will be required.

The final assessment of cardiovascular and autonomous variables will be obtained during the last 15 minutes. The rating of perceived exertion will be asked (Borg-CR10) whereas a VAS (0 denoting no sensation at all; 10 = indicating discomfort or pain onset) will be used to rate the tolerance to ES. The possible side-effects will be also checked.

The participants will be asked upon completion of the session their presumed group assignment.

Adverse effects will be checked again 48 h after finishing the experimental session.

The ES and the acquisition of the data will be done by physical therapists, but analysis of the main variables will be done by a cardiologist with the assistance of one of the physical therapists with specific knowledge in the area. The data will be introduced by a third researcher, and accuracy of the data will be checked selecting 50% of the data from each arm, by random software. If some mistakes will emerge, then the complete check will be done. The traditional statistical analysis will be reinforced by Bayesian inferences.

This is a pilot study. The objective is to check if there are statistically significant effects from the stimulation on the variables: cardiac variability, heart rate and blood pressure, before or after an isometric exercise of pressure.

For the comparison of the results obtained in the three groups of subjects, the analysis of variance or rank test procedures will be used, based on the normality or non-normality of the data obtained.

The effect of time will be assessed through a longitudinal data analysis that takes into account the dependence. For this purpose, the repeated measures Anova or Friedman's test will be used, in the parametric or non-parametric assumption, as a global comparison procedure. Possible interactions and dependence relationships between measured variables and time will be studied by means of linear regression analysis, generalised linear and/or generalised kernel-type non-parametric modelling.

Comparisons between groups (Anova or its non-parametric equivalent) will be complemented by means of their equivalents in Bayesian analysis, in order to reinforce the information provided by the p-values obtained in each procedure. For this purpose, the corresponding Bayes factors (Kass and Raftery, 1995) and the posteriori distributions of the estimated parameters will be calculated, thus increasing the precision of the confidence intervals.

Inclusion Criteria:

• University students and academic or administrative staff, non smokers, self-perceived as healthy

Exclusion Criteria:

• No history of neurological, neurosurgical, psychiatric, cardiological disease, pulmonary disease, syncope, seizure, head trauma, cranial or neck surgery, aneurisms, or any condition or treatment which could affect the functioning of the autonomous nerve system or the cardiovascular system (by self-report).
• BMI of 30 or over
• High blood pressure (systolic blood pressure of 140 mmHg or over, or diastolic blood pressure of 90 mmHg or over)
• Spontaneous respiratory rate under 10 breaths per minute
• Professional athletes
• Inability to reach the minimum amplitude required during electrical stimulation
• Any arm or hand pain (dominant side)
• Any contraindication for electrical stimulation (pregnancy or risk or being pregnant, any implanted electronical device, irritation or pain at the skin location for the electrodes, intolerance to electrical stimulation) or any condition which could affect the placement of the electrodes or the perception of the electrical stimulus.

Allocation concealment, stratified by sex, will be ensured by the use of sealed envelopes containing the treatment allocation