Article Acute Relaxation Response Induced by Tibetan Singing Bowl Sounds: A Randomized Controlled Trial Cristobal Rio-Alamos 1, *, Rodrigo Montefusco-Siegmund 2,3 , Toni Cañete 4 , Joaquín Sotomayor 1 and Alberto Fernandez-Teruel 4 1 Faculty of Medicine, Department of Psychology, Austral University of Chile, Valdivia 5090000, Chile 2 Human Cognitive Neurophysiology and Behavior Lab, Locomotor Apparatus and Rehabilitation Institute, Faculty of Medicine, Department of Kinesiology, Austral University of Chile, Valdivia 5090000, Chile 3 Centro Interdisciplinario de Estudios del Sistema Nervioso, Austral University of Chile, Valdivia 5090000, Chile 4 Medical Psychology Unit, Department of Psychiatry and Forensic Medicine, Institute of Neurosciences, Autonomous University of Barcelona, 08193 Barcelona, Spain * Correspondence: cristobal.delrio@uach.cl Abstract: The prevalence of anxiety has increased dramatically due to COVID-19, so effective preven- tive interventions are welcome. The main objective of our study was to compare the acute relaxation response (RR) induced by Tibetan singing bowl (TSB) sound-based treatment against progressive muscle relaxation (PMR) and a control waiting list group (CWL) in a single treatment session in an adult nonclinical anxious population. In this cross-sectional randomized control trial, 50 participants selected based on high state anxiety were randomly assigned to one of the experimental groups. Pre/post self-reported anxiety, electroencephalographic activity (EEG), and heart rate variability (HRV) were recorded at baseline (T1), minute 15 (T2), minute 30 (T3), and minute 45 (T4). The TSB group showed significant reductions in alpha power (from T2 to T4) and increased HRV (from T3 to T4) compared with the PMR and CWL groups. Moreover, TSB and PMR both showed significant Citation: Rio-Alamos, C.; reductions in self-reported anxiety compared with CWL, with this effect being more evident in Montefusco-Siegmund, R.; Cañete, T.; the TSB group. We concluded that a single session of TSB treatment was able to induce a more Sotomayor, J.; Fernandez-Teruel, A. evident psychological/physiological relaxation response compared with PMR and CWL. TSB could Acute Relaxation Response Induced be a relevant acute intervention in stressful situations or crisis intervention and while waiting for by Tibetan Singing Bowl Sounds: A conventional interventions. Randomized Controlled Trial. Eur. J. Investig. Health Psychol. Educ. 2023, Keywords: anxiety; music therapy; Tibetan singing bowl; heart rate variability; relaxation response 13, 317–330. https://doi.org/ 10.3390/ejihpe13020024 Academic Editors: Annie Heiderscheit and África Martos 1. Introduction Martínez Anxiety is considered one of the major mental health disorders and represents a highly Received: 13 November 2022 relevant societal problem. It is usually defined as a complex phenomenon characterized Revised: 22 January 2023 by a state of high arousal and negative valence, often leading to enhanced vigilance in the Accepted: 26 January 2023 absence of an immediate threatening stimulus [1–4]. The prevalence of anxiety has been Published: 29 January 2023 estimated at 7.3% of the worldwide population [5]. However, the coronavirus disease 2019 (COVID-19) pandemic significantly impacted mental health conditions, raising anxiety prevalence over three times, reaching a 25% prevalence for the general population [6,7]. In Chile, the prevalence for risk of anxiety has been estimated to be 39.2% of the population. Copyright: © 2023 by the authors. In this context, effective therapeutic strategies aiming to reduce anxiety/stress responses Licensee MDPI, Basel, Switzerland. are of great relevance. For this reason, despite advances in pharmacological treatments and This article is an open access article psychotherapy, innovative and preventive approaches are still required, mainly because it distributed under the terms and conditions of the Creative Commons is known that a significant proportion of patients do not respond adequately to pharma- Attribution (CC BY) license (https:// cotherapy, which can be associated with undesirable side effects (e.g., sexual dysfunction, creativecommons.org/licenses/by/ excessive perspiration, drowsiness, and weight gain) [8–11]. 4.0/). Eur. J. Investig. Health Psychol. Educ. 2023, 13, 317–330. https://doi.org/10.3390/ejihpe13020024 https://www.mdpi.com/journal/ejihpe Eur. J. Investig. Health Psychol. Educ. 2023, 13 318 Regarding effective complementary interventions, it has been proposed that treat- ments that are able to induce a relaxation response (RR) would be valuable tools for anxious patients [12–15]. Herbert Benson, the first to describe RR, defined it as the physiological counterpart of the stress response [12,13,16]. RR is known as a set of integrated physio- logical mechanisms and adjustments that can be elicited through mind–body techniques, aiming to restore the activity imbalance between the sympathetic and parasympathetic nervous systems (SNS and PNS, respectively) [12,15,16]. Thus, it has been proposed that RR inhibits SNS activity, counteracting the stress response by activating the PNS [15,16]. Hence, a more active PNS appears to be a key feature for anxiety/stress modulation. Previous studies lend support to the above contention, having shown several PNS-like responses elicited by RR techniques. These include decreased oxygen consumption, lower heart rate and blood pressure, and reductions in HPA-related hormone levels, self-reported anxiety, and neuroelectric alpha power band (8–12 Hz), as well as increased heart rate variability (HRV), among other psychological/physiological responses [14,15,17–22]. Although RR can be elicited through many different techniques (e.g., autogenic train- ing, meditation, mindfulness-based programs, etc.), Jacobson’s Progressive Muscle Relax- ation (PMR) is considered a kind of theoretical cornerstone for RR acquisition [15,21,23,24]. The effects of PMR have been thoroughly described elsewhere, but some relevant out- comes reported include better control of anxiety [25–29], reductions in perceived anxi- ety/stress [17,30,31], and reductions in cortisol and salivary alpha-amylase levels [17,27–29]. However, despite the validity acquired by PMR treatment on parasympathetic activation, some limitations have been pointed out, especially when used clinically: (i) PMR requires expert direction; (ii) it demands an active role by the patient; and (iii) it is “not so fun”, thereby decreasing treatment adherence. On the other hand, one promising complementary treatment is to use the relaxing sounds of Tibetan singing bowl (TSB) sound-based treatment. TSB consists of metal alloys used originally by Tibetan monks for meditative ceremonies [17,31–35]. The sound consists of a base tone that merges with a series of overtones, producing a continuous relaxing sound. Evidence suggests that TSB can induce improvements in distress, anxiety, depression, fatigue, and tension, as well as improvements in blood pressure, heart rate, respiratory rate, peripheral capillary oxygen saturation, cutaneous conductance, and alpha power measured by electroencephalography (EEG) [17,31,32,34]. Interestingly, a study compared monochord sound treatment, which shares the same principles as TSB treatment (a base tone merged with overtones producing a relaxing continuous sound), with PMR on self-reported anxiety and EEG. The results showed that both monochord sound treatment and PMR were able to induce RR, with the former being more effective [31]. Moreover, TSB has also been used in preoperative anxiety conditions, showing improvements in hormonal responses and self-reported anxiety measurements when compared with a control group. Specifically, TSB was able to induce: (i) reductions in self-reported anxiety; (ii) reductions in alpha-amylase levels; (iii) increases in high frequency of HRV; and (iv) increases in theta power band activity (3.5–7.5 Hz) and reductions in beta power band activity [17,31,32]. We were interested in examining the efficacy of TSB using a cross-sectional design as a starting point, evaluating the potential benefits of TSB used in an occasionally anxious (i.e., nonclinical) population. The results of acute nonpharmacological anxiety-related interventions (e.g., music, aromatherapy, massage, and acupuncture) reported by systematic reviews have shown significant positive effects of music as an effective intervention [36–38]. Similar to our study, a cross-sectional study showed that music and PMR were able to reduce anxiety after a one-day treatment session [39]. Thus, the aim of our study was to compare the potential acute RR effects induced by TSB treatment against PMR and a control waiting list (CWL) over a single treatment session. To achieve this aim, self-reported anxiety, HRV, and neuroelectrical activity (EEG) were obtained from all participants. We hypothesized that both TSB and PMR would be able to induce RR effects, with the results being more evident in the TSB group. pandemic. All sanitary safeguards were applied. The study protocol followed the Consol- idated Standards of Reporting Trials (CONSORT) criteria, as shown in Figure 1. For selection criteria, all participants signed a written consent form and completed the Spanish version of Spielberger State Anxiety Inventory (SAI) and a questionnaire about Eur. J. Investig. Health Psychol. Educ. 2023, 13 their physical condition. Selected participants (>40 in SAI) were randomly assigned 319 to one of three experimental groups: TSB, PMR, or CWL. A final sample of 50 subjects was recruited (TSB = 16, PMR = 19, CWL = 15; n/per group). The exclusion criteria were previ- ous experience with TSB or PMR, regular practice of relaxation techniques, and being un- 2. Materials and Methods der current psychiatric pharmacotherapy. A total of 50 participants were recruited (26♂ 2.1. Design and Participants and 24♀: TSB (n = 16): 9 ♂/7 ♀; PMR (n = 19): 10 ♂/9 ♀; CWL (n = 15): 7 ♂/8 ♀). Regarding their This single-blind education prospective level, 30 randomized participants (60%) were control trial current was conducted university during students, and periods 20 par- ticipants (40%) had finished their university studies and were currently working. No pan- of remission of the quarantine imposed by local governments regarding the COVID-19 sig- demic. All nificant sanitary safeguards differences were foundwere(TSBapplied. = 26.2 ±The 1.5; study PMR =protocol 26.3 ± 1;followed the Consolidated CWL = 25.9 ± 1). Standards of Reporting Trials (CONSORT) criteria, as shown in Figure 1. Figure 1. Figure Study flow 1. Study flow diagram. diagram. For selection criteria, all participants signed a written consent form and completed the Spanish version of Spielberger State Anxiety Inventory (SAI) and a questionnaire about their physical condition. Selected participants (>40 in SAI) were randomly assigned to one of three experimental groups: TSB, PMR, or CWL. A final sample of 50 subjects was recruited (TSB = 16, PMR = 19, CWL = 15; n/per group). The exclusion criteria were previous experience with TSB or PMR, regular practice of relaxation techniques, and being under current psychiatric pharmacotherapy. A total of 50 participants were recruited (26 ♂and 24 ♀: TSB (n = 16): 9 ♂/7 ♀; PMR (n = 19): 10 ♂/9 ♀; CWL (n = 15): 7 ♂/8 ♀). Regarding their education level, 30 participants (60%) were current university students, and 20 participants (40%) had finished their university studies and were currently working. No significant differences were found (TSB = 26.2 ± 1.5; PMR = 26.3 ± 1; CWL = 25.9 ± 1). 2.2. Outcome Measures 2.2.1. Heart Rate Variability (HRV) HRV recordings were obtained using a Polar H10 heart rate sensor device located at the chest and connected via Bluetooth to EliteHRV software. Once recordings were 2.2. Outcome Measures 2.2.1. Heart Rate Variability (HRV) Eur. J. Investig. Health Psychol. Educ. 2023, 13 320 HRV recordings were obtained using a Polar H10 heart rate sensor device located at the chest and connected via Bluetooth to EliteHRV software. Once recordings were fin- ished, data were transferred to a computer, and analyses were performed using Kubios finished, HRV data(Kubios software were transferred to a 3.4.3) HRV, version computer, and analyses [40]. HRV were removed artifacts were performed using before Ku- data bios HRV software (Kubios HRV, version 3.4.3) [40]. HRV artifacts were removed analyses using an automatic beat correction algorithm and the threshold-based beat cor- before data analyses using an automatic beat correction algorithm and the threshold-based beat rection algorithm provided by Kubios software [40]. Data were analyzed considering two correction algorithm provided by Kubios software [40]. Data were analyzed considering HRV dimensions (time and frequency) related to vagal tone activity. The HRV time do- two HRV dimensions (time and frequency) related to vagal tone activity. The HRV time main variables were: (i) the root mean square of successive heartbeat interval differences domain variables were: (i) the root mean square of successive heartbeat interval differences (RMSSD) and (ii) the percentage of successive heartbeat intervals that differed by more (RMSSD) and (ii) the percentage of successive heartbeat intervals that differed by more than 50 milliseconds (pNN50) [22,41]. The HRV frequency domain included: (i) VLF: very than 50 milliseconds (pNN50) [22,41]. The HRV frequency domain included: (i) VLF: very low frequency, ranging from 0 to 0.4 Hz; (ii) LF: low frequency from 0.04 to 0.15 Hz; and low frequency, ranging from 0 to 0.4 Hz; (ii) LF: low frequency from 0.04 to 0.15 Hz; and (iii) HF: high frequency from 0.15 to 0.4 Hz. Here we focused on HF, as it is considered a (iii) HF: high frequency from 0.15 to 0.4 Hz. Here we focused on HF, as it is considered a measure of parasympathetic activity only [41]. measure of parasympathetic activity only [41]. 2.2.2. 2.2.2.Electroencephalography Electroencephalography(EEG) (EEG) EEG EEG measurements were recordedand measurements were recorded andsaved savedfor foroff-line off-lineanalysis analysisusing usingaa32-channel 32-channel Emotiv Emotiv Epoc Flex gel-based sensor device connected via USB to EmotivSoftware Epoc Flex gel-based sensor device connected via USB to Emotiv Pro (ver- Pro Software sion 2.6.4) (see Figure 2 for channel locations). The EEG signals were digitized (version 2.6.4) (see Figure 2 for channel locations). The EEG signals were digitized at at a sam- pling rate of rate a sampling 128 Hz. of 128Each Hz.electrode impedance Each electrode was kept impedance wasbelow kept5below k. The EEGThe 5 kΩ. signals EEG were re-referenced signals off-line off-line were re-referenced to the algebraic mean ofmean to the algebraic the two earlobe of the electrodes. two earlobe EEG anal- electrodes. EEG yses werewere analyses performed performedusing Matlab using (Mathworks) Matlab (Mathworks) andandthethe open-source open-sourcefieldtrip fieldtripMatlab Matlab toolbox toolbox(http://www.ru.nl/fcdonders/fieldtrip/, (http://www.ru.nl/fcdonders/fieldtrip/, (accessed on 1 November (accessed on 1 November 2022))2022)) [42]. The [42]. spectral content of the frequency bands was calculated using a Hanning The spectral content of the frequency bands was calculated using a Hanning taper beforetaper before Fou- rier transformation. Fourier transformation.The The meanmeanspectral power spectral powerwaswascalculated calculated forforstandard standardfrequency frequency bands: bands:theta theta(3.5–7.5 (3.5–7.5Hz), Hz),alpha alpha(8–12 (8–12Hz), Hz),and andbeta beta(12.5–29.5 (12.5–29.5Hz).Hz). Figure 2. EEG channel allocation with a 10–20 system map (active channels in black circles). Figure 2. EEG channel allocation with a 10–20 system map (active channels in black circles). 2.2.3. Spielberg’s State Anxiety Inventory (SAI) 2.2.3. Spielberg’s State Anxiety Inventory (SAI) The Spanish version of Spielberg’s State Anxiety Inventory (SAI) is a subscale of the StateThe Spanish Trait version Anxiety of Spielberg’s Inventory Stateassesses (STAI), which Anxietythe Inventory (SAI)ofisanxiety. current state a subscale of the It consists State of 20Trait Anxiety items, with aInventory (STAI), total score which ranging assesses from the current 20 (almost never)state of(almost to 80 anxiety.always). It consistsA ofhigher 20 items, scorewith a totalgreater indicates score ranging froma 20 anxiety, and (almost cut-off never) point to 80 of 39–40 has(almost always). Ato been suggested higher score indicates greater anxiety, and detect clinically significant symptoms [31,43]. a cut-off point of 39–40 has been suggested to detect clinically significant symptoms [31,43]. 2.3. Interventions 2.3.1. 2.3. Tibetan Singing Bowl (TSB) Interventions TSB sessions 2.3.1. Tibetan were Singing performed Bowl (TSB) live by an expert (i.e., a trained music therapist). Four types TSBof sessions TSB werewere used. For accurate performed liveidentification by an expert of thea frequency (i.e., sound trained music of each TSB, therapist). Foura professional recording session was conducted in an anechoic chamber (Acoustic types of TSB were used. For accurate identification of the frequency sound of each TSB, aInstitute of the Austral University of Chile). Recordings were analyzed with Audacity software (version 3.0.2.0) (see Table 1 for TSB specifications). Each TSB session was played the same, whereby higher sound frequencies were first delivered, followed by the progressive appearance of lower sound frequencies. The TSB sound was elicited by striking the TSBs and by rubbing them with a wooden mallet. Eur. J. Investig. Health Psychol. Educ. 2023, 13 321 Table 1. Description of the diameter and frequency of tone and overtones of the four Tibetan singing bowls used. Tibetan Singing Bowl Tone Frequency 3º Overtone 4º Overtone 1º Overtone (Hz) 2º Overtone (Hz) nº-(Diameter) (Hz) (Hz) (Hz) 1-(23 cm) 99 263 465 737 971 2-(15 cm) 219 603 1115 1713 2515 3-(12 cm) 465 1185 2224 3471 4865 4-(8 cm) 927 1879 2401 4437 - 2.3.2. Jacobson’s Progressive Muscle Relaxation (PMR) PMR instructions were provided using a Bose Bluetooth Soundlink Revolve portable black speaker. The PMR session was recorded and edited in a professional recording studio in such a way that it was synchronized with the silent periods of the TSB group. Participants were instructed about tensing and relaxing the body muscles with a bottom-up procedure (i.e., beginning with the feet, followed by the legs, abdomen, chest, hands, shoulders, neck, face, and head) [44]. 2.3.3. Control Waiting List (CWL) Participants in the CWL group remained silent throughout the session, and primary and secondary outcomes were recorded exactly the same as in the TSB and PMR groups. 2.4. Procedure All experimental procedures were performed between 10:00 and 13:00. The experi- mental room was kept at 22 ± 2 ◦ C, and all recordings were obtained individually. Once they arrived, the participants were asked to sit in a comfortable armchair for HRV-EEG preparation. The SAI-1 score was obtained as both a selection criterion and basal self- reported anxiety (pre-session). Once the devices were set up properly, the experimental session began with a continuous eyes-closed EEG-HRV recording for 50 min. Within the experimental session, four [4] silent periods were included for data analysis: from 0 to 5 min (T1, baseline); from 15 to 20 min (T2); from 30 to 35 min (T3); and from 45 to 50 min (T4, post-session). Once the HRV-EEG devices were removed, the participants were asked to complete SAI-2 (post-session). Ethical approval was provided by the Ethical Committee of Bioethics on Human Research of the Austral University of Chile (Ethical Committee approval nº 0041/19) in November 2019. The clinical protocol was reviewed and approved by ISRCTN, with clinical trial registration number ISRCTN5397685. 2.5. Statistical Analyses Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, version 17, IBM Corp.) and Matlab (Mathworks) software. Appropriate repeated measures ANOVA with “SAI-1/SAI-2” and “T1 to T4 EEG/HRV” as the within-subject factor and “treatment group” as the between-subject factor was performed. One-way ANOVA was applied to all dependent variables at each time interval (EEG/HRV T1-T4). The SAI-R was obtained by calculating the difference between SAI-1 and SAI-2. Reductions in alpha activity were obtained by calculating the difference between T1 and T2, T1 to T3, and T1 to T4. Then, one-way ANOVA was applied to the SAI-R and alpha reduction computed variables. Post-hoc Duncan’s multiple range test was applied to all dependent variables following significant ANOVA effects. Pearson’s correlation coefficients were performed among the main variables. Obliquely rotated factor analysis (direct oblimin) was performed with the main variables recorded. The significance level was set at p ≤ 0.05. All variables were normally distributed. Eur. J. Investig. Health Psychol. Educ. 2023, 13 322 3. Results No differences were observed between the groups in all variables at T1. The main statistical effects between the experimental groups among all dependent variables are described in Table 2 and Figures 3 and 4. Table 2. Mean and SEM of all dependent variables with their statistical effects. ANOVA Repeated Mean ± (SEM) One-Way ANOVA Measures Dependent TSB PMR CWL “Effect” (F) * p variable (n = 16) (n = 19) (n = 15) (F) * p RMSSD (T1) 35.2 (3.55) 36.19 (3.04) 34.81 (3.63) “Treatment” RMSSD (T2) 36.54 (5.17) 32 (2.20) 30.5 (2.50) (3.6) * 0.04 “Time × treatment” RMSSD (T3) 48.4 (5.81) 34.07 (2.04) 29.4 (2.30) (4.2) * 0.001 (8.0) * 0.001 RMSSD (T4) 42.02 (2.78) 37.12 (2.76) 28.3 (2.65) Figure 3A (6.5) * 0.003 pNN50 (T1) 14.7 (2.69) 12.05 (2.33) 19.90 (3.4) pNN50 (T2) 16.5 (3.7) 8.84 (2.30) 10.8 (2.2) “Time × treatment” pNN50 (T3) 29.05 (5.55) 10.2 (2.73) 16.74 (4.77) (5.2) * 0.001 pNN50 (T4) 16.94 (2.06) 16.16 (3.04) 14.62 (4.4) HRV-HF(log) (T1) 6.3 (0.11) 6.21 (0.07) 6.14 (0.15) “Treatment” HRV-HF(log) (T2) 6.51 (0.3) 6.28 (0.47) 6.07 (0.08) (7.4) * 0.002 HRV-HF(log) (T3) 6.87 (0.23) 6.16 (0.22) 6.08 (0.14) (8.5) * 0.001 Figure 3B HRV-HF(log) (T4) 6.76 (0.22) 6.33 (0.15) 6.14 (0.12) (8.3) * 0.001 Alpha (T1) 0.01 (0.0013) 0.009 (0.0011) 0.007 (0.0012) “Time × treatment” Alpha (T2) 0.007 (0.0013) 0.008 (0.0013) 0.008 (0.0014) (4.9) * 0.001 Alpha (T3) 0.006 (0.001) 0.007 (0.0011) 0.008 (0.0016) Figure 3C Alpha (T3) 0.006 (0.001) 0.007 (0.0011) 0.008 (0.0012) Alpha (T1–T2) 0.0028 (0.0005) 0.0011 (0.0007) 0.0013 (0.0008) (3.9) * 0.03 Alpha (T1–T3) 0.0039 (0.0006) 0.002 (0.0006) 0.0003 (0.0006) (8.0) * 0.001 (8.4) * 0.001 Alpha (T1–T4) 0.0034 (0.0005) 0.0019 (0.0006) 0.0001 (0.0005) Figure 3D SAI-1 44.94 (1.1) 47.3 (1.7) 46.3 (0.82) “Treatment” (9.2) * 0.001 “Time × treatment” (11.61) * 0.001 SAI-2 35.37 (0.27) 40.0 (0.78) 44.8 (0.80) Figure 3E (45.9) * 0.001 (11.6) * 0.001 SAI-R 9.56 (1.1) 7.68 (1.3) 1.73 (0.85) Figure 3F Figure 3A–F mean and SEM of all main dependent variable effects. Figure 3E (11.6) * SAI-R 9.56 (1.1) 7.68 (1.3) 1.73 (0.85) 0.001 Figure 3F Eur. J. Investig. Health Psychol. Educ. 2023, 13 323 Figure 3A–F mean and SEM of all main dependent variable effects. (A) (B) (C) (D) (E) (F) Figure Figure 3. 3.Mean Mean±±SEM SEMofof(A)(A)Root Rootmean meansquare squareofofsuccessive successiveheartbeat heartbeatinterval intervaldifferences differences(RMSSD) (RMSSD) trajectory indicating “treatment” and “treatment x time” effects, as the TSB group trajectory indicating “treatment” and “treatment x time” effects, as the TSB group showed higher showed higher RMSSD than the PMR and CWL groups, especially in T3 and T4. (B) Trajectory of the HRV high- RMSSD than the PMR and CWL groups, especially in T3 and T4. (B) Trajectory of the HRV high- frequency(log) indicating a “treatment” effect, as overall, significant differences between TSB and frequency(log) indicating a “treatment” effect, as overall, significant differences between TSB and PMR/CWL were observed, especially at T3 and T4. (C) Occipital alpha band power along the session PMR/CWLa were indicating “timeobserved, x treatmentespecially effect”,atas T3the andTSB T4. (C) Occipital group showed alpha band power a significant along the reduction insession alpha indicating a “time x treatment effect”, as the TSB group showed a significant power (T3) in comparison with the PMR/CWL groups (D) Reduction of alpha power between reduction in alpha power T1 (T3) T2, and in comparison withT1 T1 and T3, and theand PMR/CWL groups T4, indicating (D) Reduction a significant of alpha reduction of power between occipital T1 and alpha band T2, T1 activity between TSB T1 and T3, and and CWL and T4,across the whole indicating session reduction a significant and against of PMR at T3. occipital (E) SAI-1 alpha band and SAI-2 activity scores between showing SAI reductions in TSB and PMR against CWL. (F) SAI reductions TSB and CWL across the whole session and against PMR at T3. (E) SAI-1 and SAI-2 scores showing (SAI-R), indicating a greater SAI reduction in both TSB and PMR against CWL; this effect was more SAI reductions in TSB and PMR against CWL. (F) SAI reductions (SAI-R), indicating a greater SAI evident in the TSB reduction in both TSB and PMR against CWL; this effect was more evident in the TSB group. Total sample n = 50 (TSB:16/PMR:19/CWL:15); * p < 0.05 between groups indicated (Duncan’s multiple range tests following significant ANOVA effects). Group symbols: TSB, Tibetan singing bowl; PMR, progressive muscle relaxation; CWL, control waiting list. RMSSD (T3) 0.86 - % of variance (cumulative) 37.18 59.57 Correlation −0.13 N sample = 27 Eur. J. Investig. Health Psychol. Educ. 2023, 13 324 Figure 4. EEG brain map activity for the alpha band over the experimental session. Group symbols: TSB, Tibetan Figure 4. EEGsinging bowl; brain map PMR,for activity progressive muscle the alpha band relaxation; over CWL, control the experimental waiting session. Grouplist. Total symbols: sample TSB, n = 50singing Tibetan (TSB:16/PMR:19/CWL:15). bowl; PMR, progressive muscle relaxation; CWL, control waiting list. Total sample n = 50 (TSB:16/PMR:19/CWL:15). Pearson’s correlation analyses between the main variables (EEG/HRV T1–T4; SAI-1, SAI-2, SAI-R, and alpha reduction) showed moderate negative associations between SAI-2 4. Discussion and allThealpha activity primary reduction purpose = −0.30 of this(rstudy wastoto−compare 0.37, thethe lower the SAI-2, potential acutethe higher the relaxation re- alpha reduction). SAI-2 also showed moderate negative associations with RMSSD at T3 sponse (RR) induced by Tibetan singing bowl (TSB) sound-based treatment with Jacob- and T4 (r = −0.039 to −0.41). The same negative associations were observed between SAI-2 son’s progressive muscle relaxation (PMR) and a control waiting list group (CWL) on and HRV-HF(log) (r = −0.029 to −0.033, the lower the SAI-2, the higher the RMSSD/HRV- heart rate variability (HRV), alpha power band (EEG), and self-reported anxiety in a HF(log)). High positive associations between RMSSD and pNN50/HRV-HF(log) were young adult nonclinical anxious population. found from T1 to T4 (r = 0.40 to 0.89). As no associations were found between the HRV and The main results of our study are: (i) The TSB group showed differences in both HRV EEG variables, we performed obliquely rotated factor analysis (direct oblimin) with the time/frequency domain related to parasympathetic activation. Specifically, the TSB group main theoretical and significant variables. The results identified two independent factors showed higher RMSSD and HRV high frequency (HRV-HF(log)) at T3 and T4 compared (see Table 3). with the PMR and CWL groups; (ii) the TSB group showed significant reductions in the alpha3.power Table Factorband on EEG analysis. compared Loadings ≥ 0.40 with the PMR and CWL groups (from T2 to T4); and are shown. (iii) the TSB and PMR groups showed significant reductions in self-reported anxiety (SAI) compared with the CWL group, with thisFactor reduction 1 being more evidentFactor in the2TSB group. SAI-1 0.53 0.42 SAI-2 −0.47 0.81 Alpha (T1) - - Alpha reduction (T1–T3) - −0.82 RMSSD (T1) 0.83 - RMSSD (T3) 0.86 - % of variance (cumulative) 37.18 59.57 Correlation −0.13 N sample = 27 4. Discussion The primary purpose of this study was to compare the potential acute relaxation re- sponse (RR) induced by Tibetan singing bowl (TSB) sound-based treatment with Jacobson’s progressive muscle relaxation (PMR) and a control waiting list group (CWL) on heart rate variability (HRV), alpha power band (EEG), and self-reported anxiety in a young adult nonclinical anxious population. Eur. J. Investig. Health Psychol. Educ. 2023, 13 325 The main results of our study are: (i) The TSB group showed differences in both HRV time/frequency domain related to parasympathetic activation. Specifically, the TSB group showed higher RMSSD and HRV high frequency (HRV-HF(log)) at T3 and T4 compared with the PMR and CWL groups; (ii) the TSB group showed significant reductions in the alpha power band on EEG compared with the PMR and CWL groups (from T2 to T4); and (iii) the TSB and PMR groups showed significant reductions in self-reported anxiety (SAI) compared with the CWL group, with this reduction being more evident in the TSB group. To the best of our knowledge, this is the first time that TSB treatment has been compared with PMR and a CWL in which HRV, EEG, and self-reported anxiety were recorded altogether. Previous studies have compared TSB effects against either a control group or a PMR group, as well as between naïve versus experienced meditators, but none have included both PMR and CWL groups [15,16,21,31,34]. We found that both TSB and PMR treatments were able to promote a psychological and/or physiological RR in comparison with the CWL group, with these effects being more evident in the TSB group. It has been proposed that one key feature of RR acquisition is a more active parasym- pathetic nervous system that is able to counterbalance sympathetic activity [15,16,45–47]. HRV has been considered a valid physiologic marker related to the ability to better cope with stressful situations. HRV can measure overall nervous system activity by observing changes in the heart rate cycle, as the excitement of the sinus node controlled by the auto- nomic nervous system is responsible for heart rate [47–50]. HRV parameters (i.e., RMSSD, pNN50, and HRV-HF) have been shown to be consistently associated with anxiety disorders (e.g., panic disorder, general anxiety disorder, and post-traumatic stress disorder, among others), and increases in these HRV parameters were promoted by pharmacotherapy, psy- chotherapy, or relaxation interventions [22,41,48]. Our results are in line with previous data, showing that TSB and PMR treatment were able to increase HRV parameters, which is usually associated with higher vagal tone or parasympathetic activity (i.e., RMSSD, pNN50, and HRV-HF(log)) [22,41,50–52]. Specifically, the TSB group showed a higher RMSSD at T3 (vs. PMR) and at T3 and T4 (vs. CWL) (Figure 3A), as well as an overall increase in pNN50 vs. the PMR/CWL groups (Table 2). Moreover, higher HRV-HF(log) at T3 and T4 (vs. PMR and CWL) was also observed (Figure 3D). PMR also showed higher RMSSD vs. CWL at T4, which is a finding that was expected. The HRV trajectory might be interpreted as progressive activation of the parasympathetic nervous system as the experimental ses- sion was running. The peak of the HRV RR was at minute 30 (T3), a time point at which EEG outcomes were also more evident. Interestingly, our HRV trajectories resemble those observed in previous studies in which a single TSB treatment session of 20 or 40 min was able to promote RR indexed by HRV parameters and self-reported mood, also showing the peak of relaxation at T3 (30 min) [53,54]. These results might indicate that the time of exposure seems to be relevant for the induced RR effects; however, the impact of the sound frequency of the TSB at this moment (T3) cannot be ruled out. Future studies are needed to address this question. Regarding the EEG results, the TSB group showed a significant reduction in alpha band activity from T2 to T4 (vs. CWL) and at T3 (vs. PMR) (Figure 3E,F). Divergent and incon- clusive EEG outcomes related to anxious/relaxed states have been reported [14,31,55,56]. However, two main streams of conclusions have been validated above others. First, the “theta hypothesis” proposes that relaxed states and mental concentration (such as medita- tive states) correlate with increases in frontal midline theta rhythms, something that has also been observed with anxiolytic drugs [56–59]. On the other hand, the “alpha hypothesis” proposes that alpha oscillations are enhanced in anxious individuals, particularly in anxio- genic environments, being interpreted as a sign of greater hypervigilance and readiness of the alpha system for information processing [3,19,20,60–63], a hypothesis supported by Gray’s theory of anxiety [3,64]. Previous studies have shown that alpha oscillations are positively correlated with trait/state anxiety [19]. It has also been demonstrated that higher alpha power band activity in anxious subjects increases even more after an alarm- ing/startling stimulus [19,55,65]. Thus, whereas previous studies have shown that anxious Eur. J. Investig. Health Psychol. Educ. 2023, 13 326 subjects increase alpha power after an alarming/stressful event, along the same line, we observed the opposite effect as a consequence of RR acquisition induced by the TSB inter- vention. That is, subjects with high state anxiety showed decreased alpha activity elicited by TSB sound-based treatment as an index of relaxation in which environmental alertness seemed to cease to be so relevant. Thus, a possibility is that TSB treatment desynchronized alpha activity in such a way that subjects were more “disconnected” from the environment (i.e., reduction of hyperalertness toward the environment). This suggested relaxing effect is consistent with the experimenter’s observational report, noting that TSB participants were significantly more restful and drowsy at the end of the session, something that was clearly reflected in post-session self-reported anxiety (SAI-2). Regarding self-reported anxiety, the TSB and PMR groups showed significant reduc- tions in SAI-2 compared with the CWL group, with this effect being more evident in the TSB group. Interestingly, the TSB group’s SAI-2 scores were reduced and located below the cut-off point suggested to detect clinically significant symptoms (<40), indicating a more profound subjective relaxing state, which was expected considering the pleasant nature of this intervention as well as previous findings [17,31,32,34]. PMR also reduced SAI-2 scores but in a less evident manner. This could be because PMR needs more active and focused attention, especially when patients are unfamiliar with the technique. On the other hand, TSB is a more passive and pleasant intervention that provides an interesting tool for anxiety management. One interesting feature observed in our study is that the HRV-EEG trajectories from T1 to T4 describe a very similar and consistent progression (Figure 3A–D). In this regard, it was tempting to hypothesize associations between HRV and EEG, which was not observed. Pearson’s correlation analysis showed moderate positive associations between SAI-R and RMSSD (i.e., the higher the SAI reduction, the higher the RMSSD), as well as between SAI-R and EEG alpha reduction (at T3) (i.e., the higher the SAI-R, the higher the alpha activity reduction). This is in line with previous findings [22,47,50,52] and with the expected results (i.e., TSB’s anxiolytic effects); however, no associations were found between HRV and EEG. This could indicate that two independent mechanisms might have been involved in the observed RR. In fact, when factorial analysis was performed, two independent dimensions were identified. The first dimension grouped SAI measurements (i.e., SAI-2 and SAI-R) with RMSSD, and the second grouped SAI (as in the first dimension) with alpha activity reduction (see Table 3). However, the possibility of associations between HRV and EEG variables cannot be ruled out. The neurovisceral integration model proposes that cardiac vagal tone, as reflected by HRV, is associated with prefrontal attentional control, indicating the ability to self-regulate in a more flexible manner under stress situations [45,46,66,67]. HRV is suggested to be a good index that reflects those neural mechanisms of central nervous system/autonomic nervous system integration [47,67]. Specifically, the central autonomic network, composed of several subcortical structures, innervates the heart, first through preganglionic sympathetic and parasympathetic neurons and finally through the stellate ganglia and vagus nerve. Therefore, the interplay between sympathetic and parasympathetic outputs would be responsible for HRV. The idea is that reductions in vagally mediated cardiovascular control disinhibits sympatho-excitatory influences that are usually under chronic inhibition. Thus, our HRV results might be considered an activation of vagal tone, restoring the chronic sympathetic inhibition and promoting a better balance between the sympathetic and parasympathetic nervous systems, and through this balance, eliciting a more relaxing sensation. On the other hand, as EEG measures the electrical activity generated by large groups of neurons, it is not possible to determine specific subcortical functional activity through this technique. Thus, EEG is not the proper way to test the neurovisceral hypothesis. However, it is important to highlight the “SAI-EEG” and “SAI-HRV” associations because they indicate that anxious/relaxed states are somehow related to both neural and cardiac dimensions. In conclusion, our results show that TSB and PMR were able to promote RR (psy- chological and/or physiological) over a single treatment session, with this effect being Eur. J. Investig. Health Psychol. Educ. 2023, 13 327 more evident in the TSB group. Specifically, TSB induced increases in HRV parameters related to parasympathetic activation, as well as reductions in alpha band activity, which can be seen as a possible sign of reduced environmental hyperalertness. Moreover, both TSB and PMR reduced self-reported anxiety scores, with this effect being more evident in the TSB group. Our results are in line with previous reports that showed similar TSB relaxation-inducing effects from a single treatment session [53,54]. We believe that this acute effect is a relevant feature of TSB treatment and complements the findings reported by TSB longitudinal studies [17,31,32,34]. As TSB is a passive, relaxing, and non-effort treatment for the patient, it might be an excellent intervention for the anxious environment as well as a complementary treatment for anxiety crisis. However, future studies are still necessary in order to evaluate the extent of long-lasting vs. short-term effects, as well as whether TSB’s effects are due to “time session” (i.e., if the TSB-inducing effects depend more on the time of TSB exposure) or the “frequency tone” (i.e., the frequency of the TSB used in the procedure and at each particular interval). Author Contributions: C.R.-A., R.M.-S., T.C. and A.F.-T. conceived and designed the study. C.R.-A.; J.S. and R.M.-S. carried out the intervention and analyzed the data. C.R.-A., A.F.-T. and T.C. wrote the first draft of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Fondo Nacional de Ciencia y Tecnologia, FONDECYT Chile, through the grant Fondo Iniciación a la Investigación ref. nº 11190240 (Fondecyt Iniciacion nº 11190240) (to C.R.-A.). A.F.-T. received support from grants PSI2017-82257-P (MINECO), PID2020- 114697GB-IOO (MINECO), 2017SGR-1587, and ICREA-Academia 2013 (to A.F.-T.). Institutional Review Board Statement: Ethical approval was provided by the Ethical Committee of Bioethics on Human Research of the Austral University of Chile (Ethical Committee approval nº 0041/19) in November 2019. The clinical protocol was reviewed and approved by ISRCTN, with clinical trial registration number ISRCTN5397685. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The datasets generated during and/or analyzed during the study are available from the corresponding author on reasonable request. Acknowledgments: We want to thank Felipe Otondo and Rodrigo Torres from the Acoustic Institute of the Austral University of Chile for their valuable support with the TSB sound recordings. We would also like to thank Paula Di Nota for consulting on the English composition of the current work. Conflicts of Interest: The authors declare no conflict of interest. References 1. Calhoon, G.G.; Tye, K.M. Resolving the neural circuits of anxiety. Nat. Neurosci. 2015, 18, 1394–1404. [CrossRef] [PubMed] 2. Gray, J.A. The Neuropsychology of Anxiety: An Enquiry into the Functions into the Septo-Hippocampal System; Cambridge University Press: Cambridge, UK, 1982. 3. Gray, J.A.; McNaughton, N. The Neuropsychology of Anxiety: An Enquiry into the Functions of the Septo-Hippocampal System, 2nd ed.; Oxford University Press: Oxford, UK, 2000. 4. LeDoux, J.E. Emotion circuits in the brain. Ann. Rev. Neurosci. 2000, 23, 155–184. [CrossRef] 5. Baxter, A.J.; Scott, K.M.; Vos, T.; Whiteford, H.A. 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