Immediate effects of dry needling on the autonomic nervous system and mechanical hyperalgesia: a randomized controlled trial

International Journal of Environmental Research and Public Health Article Immediate Effects of Dry Needling on the Autonomic Nervous System and Mechanical Hyperalgesia: A Randomized Controlled Trial Irene Lázaro-Navas 1,2 , Cristina Lorenzo-Sánchez-Aguilera 2, Daniel Pecos-Martín2,3 , Jose Jesús Jiménez-Rejano 4, Marcos Jose Navarro-Santana 5, JosuéFernández-Carnero 6,7,8,9,* and Tomás Gallego-Izquierdo 2,3   Citation: Lázaro-Navas, I.; Lorenzo-Sánchez-Aguilera, C.; Pecos-Martín, D.; Jiménez-Rejano, J.J.; Navarro-Santana, M.J.; Fernández-Carnero, J.; Gallego-Izquierdo, T. Immediate Effects of Dry Needling on the Autonomic Nervous System and Mechanical Hyperalgesia: A Randomized Controlled Trial. Int. J. Environ. Res. Public Health 2021,18, 6018. https://doi.org/10.3390/ ijerph18116018 Academic Editor: Paul B. Tchounwou Received: 30 March 2021 Accepted: 28 May 2021 Published: 3 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Physical Therapy, University Hospital Ramón y Cajal, 28034 Madrid, Spain; [email protected] 2University of Alcalá, Instituto de Fisioterapia y Dolor, 28805 Madrid, Spain; [email protected] (C.L.-S.-A.); [email protected] (D.P.-M.); [email protected] (T.G.-I.) 3Department of Physical Therapy, University of Alcalá, 28805 Alcaláde Henares, Spain 4Department of Physical Therapy, Faculty of Nursing, Physiotherapy and Podology, University of Sevilla, 41009 Sevilla, Spain; [email protected] 5Rehabilitation San Fernando, Alcaláde Henares, 28801 Madrid, Spain; marcosjose.navarr[email protected] 6Department of Physical Therapy, Occupational Therapy, Rehabilitation and Physical Medicine, Rey Juan Carlos University, 28922 Madrid, Spain 7Grupo Multidisciplinar de Investigación y Tratamiento del Dolor, Grupo de Excelencia Investigadora URJC-Banco de Santander, Universidad Rey Juan Carlos, 28032 Madrid, Spain 8Paz Hospital Institute for Health Research, IdiPAZ, 28029 Madrid, Spain 9Motion in Brains Research Group, Institute of Neuroscience and Sciences of the Movement (INCIMOV), Centro Superior de Estudios Universitarios La Salle, Universidad Autónoma de Madrid, 28023 Madrid, Spain *Correspondence: [email protected]; Tel.: +34-914-888-949 Abstract: Background: Dry needling (DN) is often used for the treatment of muscle pain among physiotherapists. However, little is known about the mechanisms of action by which its effects are generated. The aim of this randomized controlled trial was to determine if the use of DN in healthy subjects activates the sympathetic nervous system, thus resulting in a decrease in pain caused by stress. Methods: Sixty-five healthy volunteer subjects were recruited from the University of Alcala, Madrid, Spain, with an age of 27.78 (SD = 8.41) years. The participants were randomly assigned to participate in a group with deep DN in the adductor pollicis muscle or a placebo needling group. The autonomic nervous system was evaluated, in addition to local and remote mechanical hyperalgesia. Results: In a comparison of the moment at which the needling intervention was carried out with the baseline, the heart rate of the dry needling group significantly increased by 20.60% (SE = 2.88), whereas that of the placebo group increased by 5.33% (SE = 2.32) (p= 0.001, d = 1.02). The pressure pain threshold showed significant differences between both groups, being significantly higher in the needling group (adductor muscle p= 0.001; d = 0.85; anterior tibialis muscle p= 0.022, d = 0.58). Conclusions: This work appears to indicate that dry needling produces an immediate activation in the sympathetic nervous system, improving local and distant mechanical hyperalgesia. Keywords: dry needling; autonomic nervous system; physiological effects; cortisol; pain physiology 1. Introduction Dry needling (DN) can be defined as “a technique in which a fine needle is used to penetrate the skin, subcutaneous tissues, and muscle, with the intent to mechanically disrupt tissue without the use of an anesthetic” [ 1 ]. DN is a safe and minimally invasive technique [ 2 ] typically used for the treatment of an assortment of neuromusculoskeletal pain syndromes [ 3 – 5 ]. In recent decades, DN has been widely used to treat muscle pain Int. J. Environ. Res. Public Health 2021,18, 6018. https://doi.org/10.3390/ijerph18116018 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021,18, 6018 2 of 18 and, more specifically, the treatment of the myofascial trigger point (MTrP). The MTrP is a “hyperirritable spot within a taut band of skeletal muscle that is painful on compression, stretch, overload, or contraction of the tissue which usually responds with a referred pain that is perceived distant from the spot” [ 6 ]. MTrPs are classified as being active (in the event of spontaneous pain) or latent (absence of spontaneous pain) [ 7 ]. The presence of MTrPs in the skeletal muscle has been associated with an impaired range of motion, muscle weakness, loss of coordination, pain, and autonomic reactions [ 7 , 8 ]. DN may have been shown to be effective in the management of pain, improving range of motion, muscle strength, and coordination [ 9 ]. The possible explanations found in the literature for the decrease in pain include the effects of dry needling at the local level (producing an interruption of spontaneous electrical activity on the taut band or local vasodilation), activation of the peripheral segmental pain inhibition (explained through Gate Control Theory), or activation of the descending pathways of pain inhibition at the central nervous system level (serotonergic and noradrenergic endogenous opioid release and conditioned modulation of pain) [ 8 ]. However, these underlying mechanisms have not yet been fully clarified and are not understood [1,9]. Another possible DN mechanism for the modulation of pain is stress-induced analgesia (SIA) [ 10 ]. SIA has been described as “a reduced nociceptive response after stress exposure, which is mediated by descending inhibitory opioid and nonopioid brain circuits” [ 11 ]. SIA is influenced by activity of the autonomous nervous system (ANS) and the hypothalamic–pituitary–adrenal (HPA) axis [ 12 , 13 ]. The ANS, prior to a stressful stimulus, quickly induces physiological changes through synaptic transmissions via two branches, the sympathetic and parasympathetic nervous systems, resulting in an increase in sympathetic nervous system (SNS) activity [ 13 ]. SNS activity is usually determined by measuring skin conductance [ 14 ], heart rate, and respiratory rate values [ 15 ]. Other therapeutic procedures have been shown to produce sympathoexcitatory changes that had therapeutic benefits for patients [ 16 ]. HPA axis activity is measured by determining the cortisol level in saliva. Cortisol is an anti-inflammatory hormone regulated by the HPA axis via feedforward and feedback loops, which is related to the modulation of nociception and stress-induced analgesia [ 17 ]. It appears that the cortisol level in saliva increases with a stressful event which, in turn, appears to be related to the SIA process [ 18 ]. Therefore, the activation of the neuroendocrine system SNS–HPA axis maintains homeostasis and produces an analgesic effect [19]. Given the nature of DN [ 20 ], which is a technique that can be considered stressful, SNS–HPA axis activity could be among the possible physiological mechanisms that explain its analgesic effect [ 9 ]. Nevertheless, to our knowledge, no research has been undertaken that explores whether the SNS–HPA axis is involved in the response to DN. The purpose of this study was to determine if the application of a DN technique results in changes in skin conductance, heart rate, temperature, breathing rate, or cortisol levels in saliva between different measurements, in addition to assessing improvements in the pressure pain threshold. We hypothesized that a DN technique would result in an activation of the SNS and HPA axis, which plays a crucial role in pain modulation. 2. Materials and Methods 2.1. Study Design A randomized controlled clinical trial of parallel groups was carried out to compare a deep DN treatment with a placebo treatment, with the aim of evaluating the effects produced on the ANS and on pain. The study was performed following the CONSORT 2010 (Consolidated Standards of Reporting Trials) [ 21 , 22 ] directives and the STRICTA (Revised Standards for Reporting Interventions in Clinical Trials of Acupuncture) [ 23 ] criteria. It was approved by the Committee of Research Ethics and Animal Experimentation of the University of Alcalá(CEIT/HU/2015/06 of 23 November 2015) and registered in the Australian New Zealand Clinical Trials Registry at http://www.anzctr.org.au/ (ACTRN12616000881437) (accessed on 29 May 2021). Int. J. Environ. Res. Public Health 2021,18, 6018 3 of 18 2.2. Participants The sample comprised healthy volunteers from the student body of different degrees and the administrative staff of the University of Alcalá. These were selected using convenience non-probability sampling. The following inclusion criteria were established: (1) 18 to 65 years of age, (2) pain-free, and (3) latent MTrP in the adductor muscle of the left thumb. Participants were excluded if: (1) acute illness was present at the time of the study; (2) any of the following were present: fibromyalgia, diabetes, cardiopathy, essential arterial hypertension, hemophilia, a neurological disease, cognitive decline, a compromised immune system (HIV, cancer, hepatitis, acute immune diseases), left upper limb lymphedema; (3) taking blood thinners; (4) pregnancy; (5) fear of needles; (6) allergic to metals (nickel or chrome); (7) had participated in a dry needling/acupuncture study in the past 6 months. The subjects who met the selection criteria were informed of the study procedure by an information sheet, and they signed a data release document and provided informed consent, according to the standards of the Declaration of Helsinki. 2.3. Randomization and Blinding Each participant was assigned a code. The subjects were also randomly assigned to receive a deep DN technique or a placebo needling technique. Those not included in the study performed a concealed randomization with the program Epidat 4.2, using a 1:1 allocation ratio through simple randomization. Both the examiner who obtained all of the outcome measures and the statistician who examined the data were blinded to the subject randomization. The subjects participating in the study were blinded to the intervention, because they were not informed about the existence of a placebo group. The physiotherapist assessed the MTrPs before knowing the intervention group to which each subject belonged. 2.4. Procedure Interventions were carried out at the School of Nursing and Physiotherapy of the University of Alcalá(Madrid, Spain). All of the subjects received one session of deep dry needling or placebo needling. Measurements were performed between 9:00 a.m. and 11:00 a.m. [ 24 ]. The temperature of the room was maintained in the range of 24–25 ◦ C, and the noise level was kept to a minimum. The subjects could neither ingest alcohol or caffeine, nor perform vigorous physical activity, on the day of the study. They were not allowed to smoke during the 2 h before the study. Moreover, they could not brush their teeth, ingest any liquids, eat solid food, or chew gum 30 min before the study. The subjects lay supine on a stretcher, with their forearms free and legs stretched out. A professional physiotherapist with experience (about 15 years) in the palpation, diagnosis, and treatment of MTrPs and myofascial pain syndrome was responsible for locating and marking the MTrPs. Thus, he was considered a qualified examiner, with a good reproducibility index (k = 0.63) [ 25 ]. Subjects were instructed to remain calm and quiet, but completely awake. A ten-minute period was predetermined for the subject to acclimate to the room conditions before beginning the recording of the physiological variables [26,27]. The physiotherapist carried out the interventions in both groups, on a latent MTrP in the adductor muscle of the left-hand thumb. The same aseptic measures pertinent to the technique were applied for all subjects in both groups. Due to the supine position on the stretcher, the vision of the subjects of the DN technique was blocked. The procedure followed in the two groups is described below. 2.4.1. Deep Dry Needling Group Deep DN was performed with disposable needles (0.25 × 0.25 mm; AGU-A1038P; Agu-Punt S.L, Barcelona, Spain) [ 28 ]. The “fast-in and fast-out” technique asserted by Hong [29,30] was employed. The needle was moved up and down in multiple direc- Int. J. Environ. Res. Public Health 2021,18, 6018 4 of 18 tions (vertically, without rotations), at approximately 1 Hz frequency for 10 s, to look for LTRs [28]. 2.4.2. Placebo Needling Group A non-penetrating, simulated DN technique with placebo needles was applied to the subjects who were randomized into this group, with a modification of the protocol developed by Tough et al. [ 31 ]. Disposable sterile needles (0.25 × 0.40 mm; DB100-2540; DongBand, AcuPrime ® , Exeter, UK) were used. These needles have a red tab and cannot be distinguished from those used in the Deep Dry Needling Group (Figure 1). They also have a spring handle that can be glided up and down, imitating the movement in and out of the skin, without genuine penetration. To achieve an effective blinding [ 32 ], all of the needles were held in the box used for the Deep Dry Needling Group. The professional pinched the MTrP of the adductor muscle of the thumb, placed the guide-tube with the needle exerting some pressure, and jabbed the needle against the skin, simulating an insertion. Next, he withdrew the guide-tube, pressing the tip of the needle with his thumb, making sure the needle did not move (to ensure it did not penetrate the skin). The needle stayed in contact with the skin each time. Then, he moved the spring handle up and down 10 times at a speed of 1 Hz, in a “sparrow pecking” movement. Each time the handle moved up and down, the pressure sensation increased, replicating the feeling of a puncture. Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 4 of 18 The physiotherapist carried out the interventions in both groups, on a latent MTrP in the adductor muscle of the left-hand thumb. The same aseptic measures pertinent to the technique were applied for all subjects in both groups. Due to the supine position on the stretcher, the vision of the subjects of the DN technique was blocked. The procedure followed in the two groups is described below. 2.4.1. Deep Dry Needling Group Deep DN was performed with disposable needles (0.25 × 0.25 mm; AGU-A1038P; Agu-Punt S.L, Barcelona, Spain) [28]. The “fast-in and fast-out” technique asserted by Hong [29,30] was employed. The needle was moved up and down in multiple directions (vertically, without rotations), at approximately 1 Hz frequency for 10 s, to look for LTRs [28]. 2.4.2. Placebo Needling Group A non-penetrating, simulated DN technique with placebo needles was applied to the subjects who were randomized into this group, with a modification of the protocol developed by Tough et al. [31]. Disposable sterile needles (0.25 × 0.40 mm; DB100-2540; DongBand, AcuPrime®, Exeter, UK) were used. These needles have a red tab and cannot be distinguished from those used in the Deep Dry Needling Group (Figure 1). They also have a spring handle that can be glided up and down, imitating the movement in and out of the skin, without genuine penetration. To achieve an effective blinding [32], all of the needles were held in the box used for the Deep Dry Needling Group. The professional pinched the MTrP of the adductor muscle of the thumb, placed the guide-tube with the needle exerting some pressure, and jabbed the needle against the skin, simulating an insertion. Next, he withdrew the guide-tube, pressing the tip of the needle with his thumb, making sure the needle did not move (to ensure it did not penetrate the skin). The needle stayed in contact with the skin each time. Then, he moved the spring handle up and down 10 times at a speed of 1 Hz, in a “sparrow pecking” movement. Each time the handle moved up and down, the pressure sensation increased, replicating the feeling of a puncture. Figure 1. The needles used with the Deep Dry Needling Group are shown on the left; the needles used with the Placebo Needling Group are shown on the right. 2.5. Outcome Measures 2.5.1. Psychological Factors Before beginning the intervention, participants completed different psychometric tests to assess their initial levels of depression (the Beck Depression Inventory II (BDI-II)) Figure 1. The needles used with the Deep Dry Needling Group are shown on the left; the needles used with the Placebo Needling Group are shown on the right. 2.5. Outcome Measures 2.5.1. Psychological Factors Before beginning the intervention, participants completed different psychometric tests to assess their initial levels of depression (the Beck Depression Inventory II (BDI-II)) [ 33 ], anxiety (the State-Trait Anxiety Inventory (STAI)) [ 34 ], and pain catastrophizing (Pain Catastrophizing Scale (PCS)) [ 35 ]. This testing was undertaken because high levels in these variables can affect ANS activity and HPA activity. 2.5.2. Autonomic Nervous System Assessments: Primary Outcomes Physiological Variables To measure the ANS response when applying a DN technique, multiparametric biofeedback equipment NeXus 10 MK-II was used (Mind Media BV; Herten, the Netherlands) [ 36 – 38 ]. Data were processed using the Biotrace software, version V2015B (Mind Media BV). The skin conductance (SC) and peripheral temperature of the skin (Temp) were registered with the sensors placed as shown in Figure 2; heart rate (HR) was registered Int. J. Environ. Res. Public Health 2021,18, 6018 5 of 18 with EKG sensors, and breathing rate (BR) with a sensor placed on the sternum with an elastic band. Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 5 of 18 [33], anxiety (the State-Trait Anxiety Inventory (STAI)) [34], and pain catastrophizing (Pain Catastrophizing Scale (PCS)) [35]. This testing was undertaken because high levels in these variables can affect ANS activity and HPA activity. 2.5.2. Autonomic Nervous System Assessments: Primary Outcomes Physiological Variables To measure the ANS response when applying a DN technique, multiparametric biofeedback equipment NeXus 10 MK-II was used (Mind Media BV; Herten, the Netherlands) [36–38]. Data were processed using the Biotrace software, version V2015B (Mind Media BV). The skin conductance (SC) and peripheral temperature of the skin (Temp) were registered with the sensors placed as shown in Figure 2; heart rate (HR) was registered with EKG sensors, and breathing rate (BR) with a sensor placed on the sternum with an elastic band. Figure 2. Placement of electrodes for SC and Temp on the right hand. Measurements for the physiological variables were collected at different moments (Figure 3): Baseline (average of the 5 min before the intervention); Dry needling (average of the 10 s of the needling technique); Post-1 (average of 1 min immediately after ending the intervention); Post-2 (average of 1 min, 9 min after ending the intervention). Figure 3. Temporal chronogram of the study. Figure 2. Placement of electrodes for SC and Temp on the right hand. Measurements for the physiological variables were collected at different moments (Figure 3): Baseline (average of the 5 min before the intervention); Dry needling (average of the 10 s of the needling technique); Post-1 (average of 1 min immediately after ending the intervention); Post-2 (average of 1 min, 9 min after ending the intervention). Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 5 of 18 [33], anxiety (the State-Trait Anxiety Inventory (STAI)) [34], and pain catastrophizing (Pain Catastrophizing Scale (PCS)) [35]. This testing was undertaken because high levels in these variables can affect ANS activity and HPA activity. 2.5.2. Autonomic Nervous System Assessments: Primary Outcomes Physiological Variables To measure the ANS response when applying a DN technique, multiparametric biofeedback equipment NeXus 10 MK-II was used (Mind Media BV; Herten, the Netherlands) [36–38]. Data were processed using the Biotrace software, version V2015B (Mind Media BV). The skin conductance (SC) and peripheral temperature of the skin (Temp) were registered with the sensors placed as shown in Figure 2; heart rate (HR) was registered with EKG sensors, and breathing rate (BR) with a sensor placed on the sternum with an elastic band. Figure 2. Placement of electrodes for SC and Temp on the right hand. Measurements for the physiological variables were collected at different moments (Figure 3): Baseline (average of the 5 min before the intervention); Dry needling (average of the 10 s of the needling technique); Post-1 (average of 1 min immediately after ending the intervention); Post-2 (average of 1 min, 9 min after ending the intervention). Figure 3. Temporal chronogram of the study. Figure 3. Temporal chronogram of the study. Cortisol Free cortisol levels in saliva were determined using the Cortisol ELISA ® kit from IBL International laboratories (Hamburg, Germany) [ 39 , 40 ]. Compared to cortisol determination in plasma, this is a simple, painless, and non-invasive method. It is also less costly, no specialized medical staffing is required, and does not produce stress when performing the vein puncture [ 41 , 42 ]. In addition, the correlation coefficient between the two methods is r > 0.9 [ 39 ]. The saliva samples were collected before the intervention and three minutes after it ended [ 24 ] (Figure 3). Cortisol levels in the saliva were analyzed in the laboratories of the Research Foundation of the University Hospital Príncipe de Asturias in Alcalá de Henares. Int. J. Environ. Res. Public Health 2021,18, 6018 6 of 18 2.5.3. Pain Assessments: Secondary Outcomes Pain Intensity The Numeric Rating Scale for Pain (NRS) was used to measure the subjective pain perceived by the participant during the needling technique performance. This is a valid and reliable measuring tool to assess pain intensity during a treatment and/or intervention [ 43 ], and presents a good correlation with the Visual Analog Scale (VAS), with an overall intraclass correlation coefficient (ICC) of >0.7 [ 44 ]. To implement this scale, the subjects were asked after the intervention (to avoid talking during the recording of the physiological measures) about the maximum pain experienced during the performed needling technique, selecting a whole number between 0 and 10 that best reflected their pain intensity (0: no pain and 10: worst imaginable pain). Pressure Pain Threshold (PPT) A Wagner Force Dial ™ model FDK 20 (Greenwich, CT, USA) manual algometer was used to measure PPT before and after performing the intervention (Figure 3) on the two points previously marked: on the latent MTrP of the adductor muscle of the left thumb (point selected to examine the implicated area) and on the most painful point on palpation of the anterior tibialis muscle, approximately at 2.5 cm lateral and at 5 cm distal to the anterior tibial tuberosity [ 45 ] (point selected as an unrelated segment area to assess descending pain inhibitory mechanisms). For this purpose, the tool was placed perpendicular to the point previously marked, and through an approximate gradual increase in speed of 1 kg/s, pressure was increased until the subject started feeling pain or discomfort (without ever reaching the maximum bearable pressure); at that point, pressure was stopped. Three measurements were taken on the same point, with a 30-s time interval between each measurement, and the average of the three measurements was analyzed [16,28,46]. This method presents high reliability (ICC = 0.91) [46]. 2.6. Sample Size A prior pilot study was conducted with 20 subjects: 10 in the experimental group and 10 in the control group. In this research, the main variable was skin conductance (SC), measured with the biofeedback Nexus 10 MK-II equipment. A repeated measures contrast was used. The size effect was 0.143; a 0.05 alpha level and a power of 0.95% were assumed, plus 15% possible loss. These assumptions generated a simple size of 65 participants in total. The statistical analysis program G*Power 3.1.9.4 was used. 2.7. Statistical Analysis Data were analyzed with the statistical package SPSS for Windows, version 26.0 (SPSS Science, Chicago, IL, USA). To study the homogeneity of the groups at baseline, Student’s t-test was used for independent samples in the quantitative variables, and the Pearson’s chi-Squared test was used for the qualitative variables. To normalize the differences between participants in the variables analyzed, the data of each time period were evaluated in terms of percentage change (% Change), using the formula employed by Perry and Green [47]. Regarding the primary outcomes of the physiological variables, a separate 2-by-4 mixed model analysis of the variance was employed to assess the effects of the intervention, for which group (deep dry needling or control) was the between-subjects variable, and time (the different measurements) the within-subjects variable. An a priori alpha level of 0.05 was set. The hypothesis of interest was the group-by-time interaction. In addition, the effect size was estimated by calculating the partial Eta2 coefficient ( η p2). The difference between the two groups in the percentage change in all measurements was compared using Student’s t-test for independent samples, or, alternatively, Welch’s t-test. Bonferroni type adjustment of significance was used. For the analysis of cortisol and PPT, a separate 2-by-2 mixed model analysis of variance was employed to assess the effects of the intervention, for which group (deep dry needling Int. J. Environ. Res. Public Health 2021,18, 6018 7 of 18 or control) was the between-subjects variable, and time (the different measurements) the within-subjects variable. An a priori alpha level of 0.05 was set. The hypothesis of interest was the group-by-time interaction. Effect size was estimated by calculating η p2. The difference between both groups in % Change at baseline and at post-test was compared using Student’s t-test for independent samples, or, alternatively, Welch’s t-test. In addition, effect size was estimated using Cohen’s d, considering “small effect size” to be between 0.2 to 0.5, “medium effect size” 0.5 to 0.8, and “large effect size” greater than 0.8. In all statistical tests, the level of significance was set at 95% (p< 0.05; two-tailed test). Finally, different correlations using Pearson’s correlation coefficient were studied. First, the values obtained from the Numeric Rating Scale for Pain (NRS) for each group are shown, in addition to the existing correlations in this scale regarding % Change between the baseline–dry needling measurement of the physiological variables and regarding % Change of cortisol. In addition, correlations between % Change of the Physiological Variables and Cortisol are shown in the different measures with the Psychological Factors. 3. Results 3.1. Participant Characteristics A total of 65 subjects, with an average age of 27.78 years (SD ± 8.41 years), of whom 33 (50.8%) were men and 32 (49.2%) were women, met the selection criteria and participated in the study between November 2016 and February 2017. All of the data were collected for analysis, as shown in the flow diagram in Figure 4. Tables 1and 2contain information about the characteristics at baseline of all of the subjects in each treatment group, confirming no statistically significant differences between the groups. It can also be observed in Table 1that the subjects did not present signs of depression, anxiety, or pain catastrophizing. Table 2 also shows the values obtained regarding pain intensity perceived during the intervention. The NRS results were significantly higher in DN group than in the placebo group. Table 1. Demographic characteristics of each treatment group. Characteristics Dry Needling Group (n= 33) Mean ±SD (Range) Placebo Group (n= 32) Mean ±SD (Range) p-Value * Age (years) 26.88 ±8.05 (19–51) 28.72 ±8.79 (19–53) 0.382 Gender †Male 16 (48.5) 17 (53.1) 0.708 ‡ Female 17 (51.5) 15 (46.9) Weight (kg) 71.28 ±12.02 (46.6–98.4) 68.08 ±16.00 (47.1–108.1) 0.366 Height (m) 1.71 ±0.06 (1.58–1.82) 1.69 ±0.08 (1.54–1.83) 0.262 BMI (kg/m2)24.29 ±3.52 (18.67–32.16) 23.66 ±4.33 (17.78–33.36) 0.522 Wake-up time 7:28 ±55.8 min (5:40–9:00) 7:36 ±52.4 min (4:30–9:00) 0.565 BDI-II 5.85 ±4.75 (0–21) 5.03 ±4.04 (0–13) 0.458 STAI State Anxiety 9.82 ±7.48 (0–32) 12.34 ±6.79 (0–29) 0.160 Trait Anxiety 12.27 ±7.47 (0–25) 13.31 ±6.87 (0–25) 0.561 PCS Rumination 2.97 ±2.21 (0–8) 2.09 ±2.94 (0–9) 0.179 Helplessness 2.61 ±2.79 (0–10) 1.66 ±2.44 (0–9) 0.150 Magnification 1.94 ±1.87 (0–6) 1.31 ±1.65 (0–7) 0.158 Total Score 7.00 ±6.17 (0–21) 5.03 ±5.69 (0–20) 0.187 * Comparison needling group versus placebo group using Student’s t-test. † Absolute frequency and category percentage n(%) are shown. ‡ Pearson’s chi-squared test was used. BMI: Body Mass Index; BDI-II: Beck Depression Inventory II; STAI: State-Trait Anxiety Inventory; PCS: Pain Catastrophizing Scale. Int. J. Environ. Res. Public Health 2021,18, 6018 8 of 18 Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 8 of 18 Figure 4. Flow diagram of patients throughout the course of the study. Table 1. Demographic characteristics of each treatment group. Characteristics Dry Needling Group (n = 33) Mean ± SD (Range) Placebo Group (n = 32) Mean ± SD (Range) p-Value * Age (years) 26.88 ± 8.05 (19–51) 28.72 ± 8.79 (19–53) 0.382 Gender † Male 16 (48.5) 17 (53.1) 0.708 ‡ Female 17 (51.5) 15 (46.9) Weight (kg) 71.28 ± 12.02 (46.6–98.4) 68.08 ± 16.00 (47.1–108.1) 0.366 Height (m) 1.71 ± 0.06 (1.58–1.82) 1.69 ± 0.08 (1.54–1.83) 0.262 BMI (kg/m2) 24.29 ± 3.52 (18.67–32.16) 23.66 ± 4.33 (17.78–33.36) 0.522 Wake-up time 7:28 ± 55.8 min (5:40–9:00) 7:36 ± 52.4 min (4:30–9:00) 0.565 BDI-II 5.85 ± 4.75 (0–21) 5.03 ± 4.04 (0–13) 0.458 Analysed (n = 32) Received allocated intervention (n = 32) Allocated to Placebo needling (n = 32) Received allocated intervention (n = 33) Analysed (n = 33) Allocated to Deep dry needling (n = 33) Allocation Volunteers assessed for eligibility Analysis Excluded ( n = 0) Randomized (n = 65) Figure 4. Flow diagram of patients throughout the course of the study. Table 2. Primary and secondary outcomes. Variables Measurement Dry Needling Group (n= 33) Placebo Group (n= 32) p-Value * Effect Size Cohen’s d SC (µs) Baseline 2.95 ±1.57 2.33 ±1.26 0.082 0.43 Needling 8.42 ±4.06 7.71 ±3.22 0.437 0.19 Post-1 7.43 ±3.43 6.01 ±2.54 0.063 0.47 Post-2 4.76 ±2.49 4.12 ±2.32 0.288 0.27 HR Baseline 65.76 ±11.29 68.39 ±10.30 0.329 0.24 Needling 78.93 ±14.61 72.40 ±16.51 0.096 0.42 Post-1 66.50 ±11.46 65.69 ±12.04 0.784 0.07 Post-2 62.96 ±10.93 65.42 ±10.08 0.349 0.23 Int. J. Environ. Res. Public Health 2021,18, 6018 9 of 18 Table 2. Cont. Variables Measurement Dry Needling Group (n= 33) Placebo Group (n= 32) p-Value * Effect Size Cohen’s d Temperature (◦C) Baseline 30.81 ±4.12 31.78 ±3.79 0.275 0.24 Needling 31.21 ±3.88 31.72 ±3.28 0.328 0.14 Post-1 31.13 ±3.82 31.64 ±3.29 0.572 0.14 Post-2 31.51 ±3.93 32.61 ±3.17 0.567 0.31 BR Baseline 15.61 ±3.48 14.03 ±4.10 0.099 0.42 Needling 21.26 ±4.18 19.44 ±4.01 0.079 0.44 Post-1 19.24 ±3.61 18.27 ±4.29 0.326 0.25 Post-2 16.52 ±3.33 15.76 ±5.14 0.478 0.18 Cortisol (µg/dL) Baseline 0.53 ±0.29 0.55 ±0.27 0.734 0.07 Post-test 0.59 ±0.33 0.57 ±0.32 0.822 0.06 PPT (Kg/cm 2 ) Thumb adductor Baseline 2.13 ±0.35 2.18 ±0.43 0.562 0.13 Post-test 2.72 ±0.39 2.37 ±0.43 0.001 0.85 Anterior Tibialis Baseline 4.96 ±0.59 5.04 ±0.64 0.630 0.13 Post-test 5.57 ±0.56 5.23 ±0.62 0.022 0.58 NRS Pain Post-test 5.87 ±2.01 1.09 ±1.17 0.001 2.89 Mean ± standard error (SE) * Comparison using Student’s t-test for independent samples. Bonferroni type adjustment was used. SC: Skin conductance; HR: Heart rate; BR: Breathing rate; PPT: Pressure pain threshold; NRS: Numeric Rating Scale for Pain. 3.2. Autonomic Nervous System Assessments: Primary Outcomes The mixed model analysis of the variance of the variable HR indicated a statistically significant group-by-time interaction (F = 9.99, p< 0.001, ηp2 = 0.137) , and a time effect (Dry Needling Group F = 50.53, p< 0.001, ηp2 = 0.612; Placebo Group F = 12.57, p< 0.001, η p2 = 0.288). In this variable, no effect was found in the group factor (F = 0.04, p= 0.845, η p2 = 0.001). As shown in Table 3, comparing the Baseline with the Needling, the % Change of the HR significantly increased in the dry needling group compared to the placebo group. Subsequently, the % Change decreased more in the placebo group at Post-1, whereas the % Change from Post-1 to Post-2 was significantly greater for the HR of the dry needling group. In the variables SC, Temp, and BR, the mixed model analysis of variance indicates no statistically significant group-by-time interaction (SC F = 0.91, p= 0.380, η p2 = 0.014; Temp F = 1.80, p= 0.173, η p2 = 0.028; BR F = 0.52, p= 0.667, η p2 = 0.008), but does indicate a time effect (SC: Dry Needling Group F = 71.88 p< 0.001, η p2 = 0.692; Placebo Group F = 72.16, p< 0.001 η p2 = 0.700/Temp: Dry Needling Group F = 3.52, p= 0.042, η p2 = 0.099; Placebo Group F = 6.84, p= 0.003, η p2 = 0.181/BR: Dry Needling Group F = 28.33, p< 0.001, ηp2 = 0.470; Placebo Group F = 25.64, p< 0.001, η p2 = 0.453). No effect was found in the group factor (SC F = 2.08, p= 0.154, η p2 = 0.032; Temp F = 0.75, p= 0.389, η p2 = 0.012; BR: F = 2.50, p= 0.119, ηp2 = 0.038). A considerable increase in SC was observed after needling compared to the baseline measurement, although no significant differences existed between the two groups. However, a greater decrease (p< 0.001) was produced in the SC values of the Placebo group ( − 21.50%, SE = 1.99) in comparison with the Dry Needling Group ( − 10.36%, SE = 1.96) in the Post-1 measurement after needling (Table 3). On the contrary, no differences were found in the variables BR and temperature between groups in the percentage change of any of the measurements performed (Table 3). Regarding cortisol, no significant group-by-time interaction was found (F = 2.07, p= 0.155, η p2 = 0.032), nor significant differences between groups (F = 0.002, p= 0.969, ηp2 < 0.001) . Notwithstanding the data, an increase of 11.27% (SE = 4.76) was produced in the Dry Needling group, with a difference existing in the time factor (F = 6.84, p= 0.013, ηp2 = 0.176) . On the contrary, cortisol increased only 1.51% (SE = 3.08) in the Placebo group, and no difference was recorded in the time factor (F = 0.64, p= 0.428, ηp2 = 0.02). Int. J. Environ. Res. Public Health 2021,18, 6018 16 of 18 17. 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