Transcranial direct current stimulation (tDCS) can be used to temporarily and reversibly modulate brain functions and is an increasingly utilized tool to investigate the relationships between brain and behavior. This study aimed to identify the effects of tDCS on vocal quality, vocal range, neuromotor functioning of the larynx and self-reported vocal effort in vocally healthy singers. This is a triple-blinded, sham-controlled, randomized clinical trial. The participants were randomized to one of the three tDCS groups (primary motor cortex, cerebellum or sham). In each group a voice training was applied at the same tDCS time. The three groups are: (i) cerebellum; (ii) primary motor cortex (M1); (iii) sham. The tDCS parameters in the active groups are anodal - 2mA for 20 minutes, at the sham group the stimuli are finished 30 secondes before starting. Volunteers passed for 10 intervention days and they were evaluated for vocal performance before the sessions, in the day 5 of intervention, the last day of intervention (day 10) and 1 month later for follow up evaluation. Voice recordings was taken to assess the following vocal parameters: vocal range profile (VRP) at weak and strong intensities; vocal quality (jitter, shimmer, irregularity, glottal-to-noise excitation ratio, CPPS,HNR, and dysphonia acoustic index) at weak, habitual, and strong intensities; laryngeal diadochokinesis, and self-reported vocal effort using the adapted Borg CR10 scale.

Data collection was conducted at the Applied Neuroscience Laboratory (LANA) of the Federal University of Pernambuco (UFPE).

This study includes vocally healthy individuals who are singers without formal music or singing training. Residents of the city of Recife and the Metropolitan Region, aged 18 to 45 years. The age range was chosen considering the period of maximum vocal efficiency To determine the sample size, a pilot study was conducted with 15 participants using the G*Power 3.1 program, considering a statistical power (β) of 80% and a significance level (α) of 5%. For this analysis, the primary vocal outcome variables (vocal range profile) was considered, and the sample size considered was 45, the largest value among the selected outcomes.

Singers without formal music training and without vocal complaints were included.

Individuals who were smokers or who presented signs and symptoms of laryngeal, pharyngeal or respiratory diseases at the time of data collection were excluded from the sample. Volunteers who were using neuroactive medications regularly, have metal implants in the cervical region or above, and had any history of seizures or epilepsy were also excluded.

The sample of singers was constituted by convenience through individuals who were singers without vocal complaints of both sexes, recruited through advertisements in electronic media. The first contact with the volunteers was through a screening to verify the eligibility criteria.

Added to tDCS is the vocal exercise, performed with the aid of a silicone tube with the following dimensions: 35cm in length, 1cm in diameter and 2mm in thickness, and with the distal end immersed at a distance of 3cm from the bottom of a 500ml plastic bottle containing 2/3 of water and with the spaces delimited. This study investigated the combined effects of tDCS and vocal exercises on singers voices. We conducted a clinical trial to examine the impact of concurrent neuromodulation and vocal training. Anodal tDCS was applied at 2mA for 20 minutes to three groups: (i) M1; (ii) Cerebellum; and (iii) sham. All groups performed vocal exercises using a flexible tube (FT) for a total of 12 minutes. Participants completed an adverse effects questionnaire after each session to report any discomfort. Vocal assessment was performed before the intervention to obtain results on vocal parameters, vocal range profile, and vocal effort, as well as on the 5th day after the start of the intervention, on the 10th day, and 1 month after the end of the activities. Voice recordings was taken to assess the following vocal parameters: vocal range profile (VRP) at weak and strong intensities; vocal quality (jitter, shimmer, irregularity, glottal-to-noise excitation ratio, CPPS,HNR, and dysphonia acoustic index) at weak, habitual, and strong intensities; laryngeal diadochokinesis, and self-reported vocal effort using the adapted Borg CR10 scale. The participants answered the Borg CR 10-BR scale questionnaire - adapted for vocal effort to assess the singers self-perception of the proposed tasks, however, the singers are instructed to report the intensity of the effort produced during the singing task of a song of their choice, according to their self-perceived difficulty.

In order to determine the volunteers who would be in each group, and for subsequent sample size calculation, a randomization was performed for 15 volunteers, by an external researcher, using a random table and adjusted to allow for counterbalancing. Codes were assigned to the groups, which were kept in opaque and numbered envelopes, available only to the person responsible for applying the tDCS. In this way, the evaluator, the therapist responsible for the vocal technique with a flexible tube immersed in water, and the singer remain blinded to the type of stimulation to which the participants was subjected. After performing the sample size calculation, a new randomization was performed to allocate the participants who continued in the data collection to complete the number of necessary volunteers.

The participants was allocated into three groups, namely: (i) M1; (ii) Cerebellum; (ii) sham. In all groups, 10 consecutive sessions of anodal tDCS associated with vocal exercise was performed, these only do not occur on weekends. For the application of tDCS in the groups: (i) M1, the stimulation electrode (anode) was positioned over C5 (international 10-20 EEG system) with the cathode (negative pole) positioned over the contralateral supraorbital region; (ii) Cerebellum, the stimulation electrode over the cerebellar cortex (inion, slightly to the right) and the reference electrode (cathode) was positioned over the right deltoid muscle; (ii) sham, the montage equivalent to cerebellar stimulation was performed, however in the sham stimulation there was an interruption of the stimulation 30 seconds after the start, thus the current intensity drops to zero mA, but the electrodes remained on the scalp of the volunteers until the end of the vocal exercise. The exercise with FT was performed for a total of 20 minutes (3 minutes of FT, four sequences, followed by 2 minutes of interval) in all groups. In the sham group, the electrodes was only removed after the end of the vocal exercises, so that the patient was unaware of the type of stimulation (real or sham) to which they were exposed. To blind the evaluator and therapist, a towel was placed over the volunteers head every day. Participants were instructed to sit comfortably while electrodes were placed on their scalp and tDCS stimulation was initiated. Following stimulation, participants performed vocal exercises using a flexible tube. The exercise involved producing sustained /u/ vowels in ascending and descending scales, tailored to each participants vocal range (soprano, mezzo-soprano, contralto, tenor, baritone, or bass). These scales were based on pre-recorded piano accompaniments.

The flexible tube was 35 cm long, 1 cm in diameter, and 2 mm thick. One end of the tube was submerged 3 cm into a 500 ml bottle filled with water. Participants produced four sets of vocal exercises, each lasting 3 minutes, with 2-minute rests between sets, totaling 20 minutes of vocal therapy. This ensured that the vocal exercises concluded simultaneously with the tDCS session. Voice recordings was taken to assess the following vocal parameters: vocal range profile (VRP) at weak and strong intensities; vocal quality (jitter, shimmer, irregularity, glottal-to-noise excitation ratio, and dysphonia acoustic index) at weak, habitual, and strong intensities; laryngeal diadochokinesis, and self-reported vocal effort using the adapted Borg CR10 scale. This research was conducted in accordance with the ethical guidelines established by Brazils National Health Council (Resolution 466/12). The study was approved by the Human Research Ethics Committee of UFPE Health Sciences Center (number 5.758.856). Participants provided written informed consent after being fully informed about the studys objectives, procedures, potential risks, and benefits.

While the study posed minimal risks, participants might experience mild side effects from tDCS, such as tingling, itching, or burning sensations at the electrode sites. These side effects were carefully monitored. Additionally, some participants might experience discomfort during vocal exercises. However, these issues were addressed promptly.

Participants benefited from receiving guidance on vocal health and a personalized flexible tube. Data collected from vocal assessment software will be stored securely for at least 5 years. All data collected using Vocalgrama and VoxMetria software were analyzed using the respective software. In Vocalgrama, the following outcome measures were extracted: semitom range, and area of the vocal range profile. In VoxMetria, the following measures were extracted: jitter, shimmer, noise-to-harmonic ratio (NHR), and GNE from a 6-second sustained /ε/ vowel, excluding the first and last seconds. Cepstral analysis was conducted (CPPS and DAI). Audio recordings were made at a sampling rate of 44 kHz and 16-bit resolution. VoxMetria was also used to count the number of peaks in diadochokinetic tasks. Perceived effort was assessed using the Borg CR 10-BR scale adapted for vocal effort.

Data normality was assessed using the Shapiro-Wilk test. The non parmetric friedman repeated mensures was applied between the times of evaluation. All analyses was performed using SPSS (version 26.0, SPSS Inc, Chicago, USA) with a significance level of 5%.

Inclusion Criteria:

• Vocally healthy singers without formal singing education

Exclusion Criteria:

• Smokers
• Abnormalities in previous screening for voice symptoms
• Exhibited signs and symptoms of laryngeal, pharyngeal, or respiratory diseases at the time of collection
• Used neuroactive medications
• Had metallic implants in the cervical region or above
• Had any history of seizures or epilepsy