Laser Auriculotherapy for Anxiety Symptoms Across Diverse Populations: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear colleagues, This study is relevant in the field of integrative and complementary therapies. It presents the main steps for a systematic review with meta-analysis. However, here are some suggestions for improvement:

1- It is important to review the section on the importance of the study for public health – “This systematic review and meta-analysis demonstrated that low-level laser auriculotherapy (LLLT-AT) was associated with a reduction in anxiety symptoms, pain intensity, and disability, compared to control interventions,” as the study is presenting only six studies with distinct populations, small samples, and high heterogeneity.

2- The study's conclusion needs to be revised, as the authors cannot conclude the benefits of laser auriculotherapy, given that the findings in the sensitivity analysis results were not significant. Furthermore, only six studies were included, with small samples and a diverse population. More caution is needed in the conclusion.

3- The results need to be discussed more comprehensively, discussing the differences between dosimetry, energy, and type of light provided by the laser. In addition, it is necessary to discuss the different controls and interventions that were associated with laser auriculotherapy. Discuss confounding bias.      



 

Author Response

1. REVIEWER 1 COMMENTS AND SUGGESTIONS

Comments to the author (if any):

Dear colleagues, this study is relevant in the field of integrative and complementary therapies. It presents the main steps for a systematic review with meta-analysis. However, here are some suggestions for improvement:

 

Dear Reviewer 1,

We thank you for your time and for the opportunity to revise our manuscript. We appreciate your constructive comments and have carefully considered each of your suggestions. Below, we provide a detailed, point-by-point response to every comment. All revisions made in response to your observations have been highlighted in blue in the revised manuscript.

(1) It is important to review the section on the importance of the study for public health – “This systematic review and meta-analysis demonstrated that low-level laser auriculotherapy (LLLT-AT) was associated with a reduction in anxiety symptoms, pain intensity, and disability, compared to control interventions,” as the study is presenting only six studies with distinct populations, small samples, and high heterogeneity.

R: We thank the reviewer for this valuable comment. We agree that the previous wording could be interpreted as overstating the strength of the available evidence. Accordingly, we revised the Public Health Significance section by replacing "demonstrated" with more cautious language ("suggests" and "may"), while explicitly acknowledging the limited number of included studies and the heterogeneity of the available evidence. We also revised the Abstract and Conclusions to ensure a more balanced interpretation of the findings.

In addition, a new subsection entitled "Public Health Implications" was incorporated into the Discussion to better contextualize the potential relevance of LLLT-AT within public health, while emphasizing that the current evidence remains preliminary and that further high-quality randomized controlled trials are required before broad clinical implementation can be recommended.

(2) The study's conclusion needs to be revised, as the authors cannot conclude the benefits of laser auriculotherapy, given that the findings in the sensitivity analysis results were not significant. Furthermore, only six studies were included, with small samples and a diverse population. More caution is needed in the conclusion.

R: We thank the reviewer for this important comment. We agree that the Conclusions should reflect the strength and limitations of the available evidence more cautiously. Accordingly, we revised the Conclusions to adopt more balanced wording, emphasizing the substantial clinical heterogeneity, the variability in intervention protocols and dosimetric parameters, and the low-to-moderate certainty of the evidence. We also moderated the interpretation of the findings throughout the manuscript, including the Public Health Significance, Public Health Implications, Abstract, and Conclusions, and reinforced that further well-designed randomized controlled trials are required to strengthen the current evidence base before more definitive conclusions can be drawn. The revised Conclusions are presented below:

• Revised Conclusion: “LLLT-AT was associated with reduced anxiety symptoms across different clinical populations, including individuals with temporomandibular disorders, perioperative anxiety, cancer-related conditions, and stress-related symptoms. Favorable effects were also observed for pain intensity and disability outcomes. In addition, the available evidence suggests that LLLT-AT may represent a safe, well-tolerated, and non-invasive alternative to other auriculotherapy approaches, demonstrating lower risks of selected local adverse effects compared with control interventions. However, substantial heterogeneity in clinical populations, intervention protocols, and dosimetric parameters, together with the low-to-moderate certainty of evidence, requires cautious interpretation of the current findings. Future high-quality RCTs are warranted to confirm the observed findings and reduce the remaining uncertainty regarding the effectiveness of LLLT-AT for anxiety-related symptoms”.

(3) The results need to be discussed more comprehensively, discussing the differences between dosimetry, energy, and type of light provided by the laser. In addition, it is necessary to discuss the different controls and interventions that were associated with laser auriculotherapy. Discuss confounding bias.

R: We thank the reviewer for this valuable comment. We expanded the Discussion to provide a more comprehensive interpretation of the findings. Specifically, we incorporated a detailed discussion of the variability in laser dosimetry (wavelength, emission mode, energy density, total delivered energy, and treatment protocols) and its potential influence on photobiomodulatory responses and between-study heterogeneity. We also expanded the discussion of the different comparator interventions and included a discussion of potential confounding related to clinical populations, intervention protocols, auricular point selection, and concomitant therapies. These revisions have been incorporated into Sections 4.3 and 4.4 of the Discussion in the revised manuscript.

We thank the reviewer once again for the valuable and constructive feedback. We believe that the revisions undertaken have substantially strengthened the manuscript and improved its methodological transparency, scientific rigor, and clinical interpretation.

Thank you for your time and consideration.

Sincere regards,

The research team

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Below you will find some observations that will undoubtedly improve the quality of the manuscript.

The manuscript addresses a clinically relevant question regarding the efficacy of low-level laser therapy auriculotherapy (LLLT-AT) on anxiety symptoms. The review adheres to PRISMA 2020 guidelines, is prospectively registered in PROSPERO, and employs well-established methodological tools (RoB 2, GRADE). The topic is pertinent to public health, given the growing interest in non-pharmacological complementary interventions for anxiety management. However, the manuscript presents substantial methodological and reporting limitations that must be addressed before it can be considered for publication.

The main concerns are: (1) substantial unexplained heterogeneity; (2) an extremely small evidence base (n = 6 RCTs, 386 participants); (3) highly heterogeneous clinical populations that challenge the validity of quantitative syntheses; (4) inconsistencies in reference numbering and statistical data reporting; and (5) insufficient emphasis on the sensitivity analysis results which showed a non-significant effect after excluding studies with high risk of bias in the discussion and conclusions.

Title

The title is appropriate and descriptive. However, it should be noted that the included population does not correspond exclusively to individuals with formally diagnosed anxiety disorders, but rather to diverse populations with anxiety symptoms across heterogeneous clinical contexts (temporomandibular disorders, cancer, perioperative care, and university students). The authors should consider whether the current wording accurately reflects the scope of the included participants.

Abstract

The abstract reports the primary pooled effect (SMD = −0.65; 95% CI: −1.17 to −0.13) as the main finding. However, it omits the sensitivity analysis result which excluded studies with high risk of bias and yielded a non-significant effect (SMD = −0.35; 95% CI: −0.94 to 0.25; p = 0.2524). This finding is fundamental to an accurate interpretation of the evidence base and must be reported in the abstract to avoid overstating the therapeutic effect of LLLT-AT.

The abstract states that the certainty of evidence ranged from ‘very low’ to ‘moderate’, but does not specify which outcomes correspond to each category. At minimum, the abstract should indicate that the primary outcome (anxiety symptom reduction) was supported by very low to low certainty evidence.

Introduction

The introduction adequately justifies the prevalence and burden of anxiety disorders. However, the transition to LLLT-AT as a specific intervention is somewhat abrupt. A more explicit synthesis of why previous reviews on conventional auriculotherapy do not adequately address LLLT-AT — citing existing systematic reviews already referenced in the manuscript (e.g., Vieira et al., 2018; de la Barra Ortiz et al., 2025) — would strengthen the rationale.

The description of the neurobiological mechanisms of LLLT (cytochrome c oxidase, ATP, reactive oxygen species) is relevant but overly extensive for the introduction section, and much of this content is repeated in the discussion (Section 4.1). The authors are encouraged to condense the mechanistic background in the introduction and refer the reader to the discussion for the detailed development of these concepts.

The stated objective (‘to evaluate the effects of laser auriculotherapy on reducing anxiety symptoms in individuals with anxiety or anxiety disorders’) is appropriate but somewhat broad. Given that the analysis also includes secondary outcomes such as pain intensity and disability, the authors should either expand the objective to include these outcomes or explicitly differentiate between primary and secondary objectives.

Methods

The eligibility criteria are generally well described. However, the inclusion of studies using LLLT-AT ‘in combination with other interventions, provided that the effects of the laser intervention could be reasonably interpreted’ is a vague formulation that introduces selection bias risk. The authors must detail how this criterion was operationalised in practice (e.g., what specific decision rule was applied to determine whether the laser’s effects were ‘reasonably interpretable’ within a combined design).

The inclusion of participants with ‘anxiety symptoms’ (without a formal diagnosis) alongside those with ‘diagnosed anxiety disorders’ generates a significant source of indirectness, which the GRADE assessment acknowledges. However, this heterogeneity is not sufficiently discussed in the Methods section. The authors should specify a priori how they planned to address this distinction in subgroup analyses, and justify why pooling these populations is methodologically appropriate.

The search is described as last updated on May 24, 2026. An inconsistency was identified: while the abstract reports this date, the PROSPERO registration date is October 1, 2025. The authors must clarify whether the updated search is aligned with the registered protocol and report any deviations or amendments made to the original protocol.

The authors state that Fleiss’ kappa was calculated for the risk of bias assessment, but the obtained value and its interpretation are not reported anywhere in the manuscript. This statistic must be explicitly reported (kappa value and interpretation according to a conventional scale) either in the Methods or in the corresponding Results subsection.

A continuity correction of 0.5 was applied for studies with zero-event cells in the dichotomous adverse event analyses. Given that the majority of included studies reported zero events in both groups for several outcomes (e.g., ear pain, hyperemia), this approach may artificially inflate risk ratio estimates. The authors must explicitly justify this choice over established alternatives — such as the Sweeting continuity transformation or exact methods — and acknowledge this as a limitation of the adverse event analyses.

The application of split-comparison procedures for the three-arm trials (Lin et al.; Marcondes et al.) is correctly justified by reference to Cochrane recommendations. However, the specific procedure used to distribute the shared control group sample between the two resulting comparisons is not described. The authors must clarify how the control group sample size was handled (e.g., halved between comparisons), in accordance with the Cochrane Handbook guidance.

Results

Table 1 contains apparent inconsistencies. First, the mean age reported for Marcondes et al. (2025) is 40.3 (16.4) years, identical to the value reported for Lin et al. (2024). Given that Marcondes et al. enrolled exclusively women with advanced breast cancer undergoing chemotherapy, this coincidence should be verified to rule out a typographical error. Second, De la Barra et al. (2026) reports 25 females and 19 males, which sums to 44, not 47 as stated as the total sample. The authors must verify and correct these figures.

The weighted risk of bias analysis is well presented. However, as noted in Comment 2.3.4, the Fleiss’ kappa value must be reported in this section. Additionally, since the first author (de la Barra Ortiz H.A.) is also the principal author of one of the included studies (reference [49]: De la Barra et al., 2026), the manuscript should explicitly state whether independent reviewers blind to authorship conducted the risk of bias assessment for this specific study, in order to ensure methodological impartiality.

The substantial heterogeneity identified (I² = 78.9%) is acknowledged but not rigorously explored through pre-specified subgroup analyses or meta-regression. The subgroup analysis by comparator type (no treatment, placebo, active control) is informative but insufficient to explain the observed variability. The authors are strongly encouraged to perform and report at least one additional subgroup analysis by clinical population type (e.g., TMD vs. perioperative anxiety vs. cancer vs. university students), as clinical context is a plausible source of heterogeneity.

The sensitivity analysis excluding studies with high risk of bias (Fernandes et al. and Lin et al.) yielded a non-significant pooled effect (SMD = −0.35; 95% CI: −0.94 to 0.25; p = 0.2524). This is the most methodologically robust estimate in the entire manuscript and should receive substantially greater prominence in the Results, Discussion, and Conclusions sections. In its current form, the manuscript prioritises the overall effect estimate (which includes high-risk-of-bias studies) over the sensitivity result, which may lead readers to overestimate the evidence for LLLT-AT’s anxiolytic effects. This must be addressed and rebalanced throughout the manuscript.

The adverse event meta-analyses are almost entirely driven by a single study (Lemos et al., 2024), as most included studies reported zero events in both groups. While the authors briefly acknowledge this in the text, they must be more explicit in stating that these pooled analyses are, in practice, single-study estimates artificially amplified by the continuity correction applied to zero cells. It is recommended that these results be presented descriptively or narratively rather than as formal meta-analyses, and that the pooled risk ratio estimates be interpreted with the utmost caution.

Table 3 contains an inconsistency in the sample sizes for the outcome ‘Disability (JFLS-8): LLLT-AT vs. Placebo’: the table reports 44 participants in the LLLT-AT group and 44 in the control group (total = 88), whereas the text reports 31 in the LLLT-AT group and 26 in the control group (total = 57). The authors must verify and correct these values to ensure internal consistency between tables and text.

Discussion

As indicated in Comment 2.4.4, the manuscript in its current form does not adequately balance the presentation of the primary pooled effect (significant) against the sensitivity analysis (non-significant). The discussion and conclusions must reflect that the evidence is, at best, preliminary and not conclusive for the anxiety outcome, given the fragility of the effect when high-risk-of-bias studies are excluded.

The neurobiological mechanisms section is well documented but contains speculative claims that are not directly supported by LLLT-AT-specific evidence. Much of the mechanistic reasoning is inferred by analogy from transcranial photobiomodulation, non-auricular vagal stimulation, or needle-based auriculotherapy studies. The authors must clearly distinguish between mechanisms with direct LLLT-AT evidence and those inferred by analogy, using appropriately cautious language (‘may’, ‘has been proposed’, ‘remains to be established’).

The discussion on clinical heterogeneity is appropriate but should more explicitly address the implications of combining populations as diverse as TMD patients, parents in a surgical waiting room, university students, and women with advanced breast cancer in a single meta-analysis. The authors should provide a more critical reflection on whether the shared denominator of ‘anxiety symptoms’ is sufficient to justify quantitative synthesis across such markedly different clinical contexts, psychosocial burdens, and comorbidity profiles.

The dosimetric considerations section is informative but employs a prescriptive, forward-looking tone that the current evidence cannot support. The parameter ranges described (wavelength 660–905 nm; energy density 0.19–4 J/cm²; treatment time 20–56 seconds per point) are so broad as to constitute a description of what has been used rather than evidence-based optimal dosimetry. The authors must use more cautious language and explicitly acknowledge that these ranges reflect the existing literature and do not constitute dosimetric recommendations.

The first and corresponding author (de la Barra Ortiz H.A.) is also the lead author of one of the six included studies (reference [49]: De la Barra et al., 2026, Int. J. Environ. Res. Public Health), which is the study with the highest PEDro score (9/10) and contributes substantially to the anxiety and disability analyses. Although this does not invalidate the review, the manuscript must explicitly address this potential conflict of interest in the Limitations section and declare whether specific methodological safeguards were implemented (e.g., independent external reviewers conducting the risk of bias assessment for study [49]).

Conclusions

The current conclusions state that ‘LLLT-AT was associated with reduced anxiety symptoms across different clinical populations’ without adequately qualifying that this effect did not hold in the most methodologically rigorous sensitivity analysis. The conclusions must be reformulated to accurately reflect the state of the evidence: the effect is preliminary, not robust to sensitivity analyses, and based on a small body of highly heterogeneous studies. The tone of the conclusions must be consistent with the very low to low GRADE certainty rating assigned to the primary outcome.

The clinical and public health implications presented in Section 4.6 and summarised in the conclusions exceed what the available evidence can support. Statements suggesting the potential integration of LLLT-AT into primary care or community-based settings should be clearly framed within the context of the available evidence quality and must not be presented as direct clinical recommendations.

Errors

The intervention column header in Table 3 uses ‘LLL-AT’ instead of ‘LLLT-AT’. This typographical error must be corrected throughout the table.

References

Throughout the manuscript, inconsistencies in reference numbering were identified. For example, in Section 3.3, the citation ‘(24–29)’ implies a consecutive range, whereas the six included studies correspond to references [24], [25], [46], [47], [48], and [49] — not a consecutive sequence. This pattern is repeated at several other points in the text. The authors must conduct a thorough review of all in-text citations to ensure consistency with the reference list.

References [7] (Wehry et al., 2015) and [8] (Creswell et al., 2014) address the assessment and management of anxiety disorders in children and adolescents, not in adults. Their use to support standard treatment approaches for adult anxiety disorders is potentially misleading. The authors should replace these with references specifically addressing adult populations.

Author Response

1. REVIEWER 2 COMMENTS AND SUGGESTIONS

Comments to the author (if any):

Dear authors, Below you will find some observations that will undoubtedly improve the quality of the manuscript.

The manuscript addresses a clinically relevant question regarding the efficacy of low-level laser therapy auriculotherapy (LLLT-AT) on anxiety symptoms. The review adheres to PRISMA 2020 guidelines, is prospectively registered in PROSPERO, and employs well-established methodological tools (RoB 2, GRADE). The topic is pertinent to public health, given the growing interest in non-pharmacological complementary interventions for anxiety management. However, the manuscript presents substantial methodological and reporting limitations that must be addressed before it can be considered for publication.

The main concerns are: (1) substantial unexplained heterogeneity; (2) an extremely small evidence base (n = 6 RCTs, 386 participants); (3) highly heterogeneous clinical populations that challenge the validity of quantitative syntheses; (4) inconsistencies in reference numbering and statistical data reporting; and (5) insufficient emphasis on the sensitivity analysis results which showed a non-significant effect after excluding studies with high risk of bias in the discussion and conclusions.

 

Dear Reviewer 2,

We sincerely thank you for your careful and thorough review of our manuscript. We greatly appreciate the time and effort devoted to providing detailed and constructive comments. Your observations have helped us improve the methodological transparency, accuracy of reporting, and balance in the interpretation of our findings. In response to your suggestions, we have substantially revised several sections of the manuscript, including the Methods, Results, Discussion, Limitations, and Conclusions, with particular attention to the interpretation of sensitivity analyses, certainty of evidence, clinical heterogeneity, adverse event analyses, and the strength of the conclusions. Below, we provide a detailed, point-by-point response to each comment. All revisions made in response to your observations have been highlighted in purple in the revised manuscript.

 

Title

(1) The title is appropriate and descriptive. However, it should be noted that the included population does not correspond exclusively to individuals with formally diagnosed anxiety disorders, but rather to diverse populations with anxiety symptoms across heterogeneous clinical contexts (temporomandibular disorders, cancer, perioperative care, and university students). The authors should consider whether the current wording accurately reflects the scope of the included participants.

R: We thank the reviewer for this thoughtful observation. We agree that the included studies evaluated anxiety symptoms across diverse populations rather than exclusively individuals with formally diagnosed anxiety disorders. Although the original title intentionally referred to anxiety symptoms (Laser auriculotherapy for anxiety symptoms: a systematic review and meta-analysis of randomized controlled trials), we have revised it to more accurately reflect the scope of the included participants.

• The revised title is: "Laser auriculotherapy for anxiety symptoms across diverse populations: a systematic review and meta-analysis of randomized controlled trials."

 

Abstract

(2) The abstract reports the primary pooled effect (SMD = −0.65; 95% CI: −1.17 to −0.13) as the main finding. However, it omits the sensitivity analysis result which excluded studies with high risk of bias and yielded a non-significant effect (SMD = −0.35; 95% CI: −0.94 to 0.25; p = 0.2524). This finding is fundamental to an accurate interpretation of the evidence base and must be reported in the abstract to avoid overstating the therapeutic effect of LLLT-AT.

The abstract states that the certainty of evidence ranged from ‘very low’ to ‘moderate’, but does not specify which outcomes correspond to each category. At minimum, the abstract should indicate that the primary outcome (anxiety symptom reduction) was supported by very low to low certainty evidence.

R: We thank the reviewer for this valuable comment. In response, we revised the Abstract to provide a more balanced and transparent summary of the evidence. Specifically, we incorporated the results of the sensitivity analysis, highlighting the non-significant effect after excluding studies with a high risk of bias. We also specified the certainty of evidence for the most relevant outcomes, clarifying that anxiety, pain intensity, and disability were supported by very low-to-low certainty evidence, whereas selected adverse events were supported by moderate certainty evidence. Finally, to better reflect the scope of the included studies, we revised the study objective to indicate that the review evaluated anxiety symptoms across diverse populations rather than exclusively individuals with anxiety disorders.

The revised Abstract now includes the following statements:

• "However, sensitivity analyses excluding studies with high risk of bias yielded a non-significant effect for anxiety outcomes (SMD = −0.35; 95% CI: −0.94 to 0.25). Interpretation of the findings should be considered cautiously due to substantial heterogeneity and the very low-to-low certainty of evidence for anxiety outcomes. Across all outcomes, the certainty of evidence ranged from very low to moderate, with very low-to-low certainty for anxiety, pain intensity, and disability outcomes, and moderate certainty for selected adverse events (ear pain, hyperemia, and itchy ear)."
• "This systematic review aimed to evaluate the effects of LLLT-AT on anxiety symptoms across diverse populations."

 

Introduction

(3) The introduction adequately justifies the prevalence and burden of anxiety disorders. However, the transition to LLLT-AT as a specific intervention is somewhat abrupt. A more

explicit synthesis of why previous reviews on conventional auriculotherapy do not adequately address LLLT-AT — citing existing systematic reviews already referenced in the manuscript (e.g., Vieira et al., 2018; de la Barra Ortiz et al., 2025) — would strengthen the rationale.

The description of the neurobiological mechanisms of LLLT (cytochrome c oxidase, ATP, reactive oxygen species) is relevant but overly extensive for the introduction section, and much of this content is repeated in the discussion (Section 4.1). The authors are encouraged to condense the mechanistic background in the introduction and refer the reader to the discussion for the detailed development of these concepts.

The stated objective (‘to evaluate the effects of laser auriculotherapy on reducing anxiety symptoms in individuals with anxiety or anxiety disorders’) is appropriate but somewhat broad. Given that the analysis also includes secondary outcomes such as pain intensity and disability, the authors should either expand the objective to include these outcomes or explicitly differentiate between primary and secondary objectives.

R: We thank the reviewer for these insightful suggestions. In response, we strengthened the rationale for the study by providing a clearer synthesis of the existing evidence on conventional auriculotherapy and highlighting the current knowledge gap regarding LLLT-AT for anxiety symptoms. Specifically, we clarified that previous systematic reviews primarily evaluated needle-, seed-, acupressure-, and electrically based auriculotherapy, whereas no comprehensive synthesis had specifically addressed laser auriculotherapy for anxiety symptoms. We also revised the study objective to better reflect the scope of the review, explicitly indicating that anxiety symptom reduction was the primary outcome, while pain intensity, disability, and adverse events were evaluated as secondary outcomes. Furthermore, the mechanistic background was condensed to avoid redundancy with the Discussion, while retaining the key neurophysiological rationale supporting the potential effects of LLLT-AT.

The revised Introduction now includes the following statements:

• "LLLT, or photobiomodulation therapy, uses red or near-infrared light to stimulate auricular acupoints in a non-invasive manner. Similar to conventional auriculotherapy, LLLT-AT is thought to modulate neural and autonomic pathways through stimulation of the auricular branches of the vagus, trigeminal, facial, and cervical nerves, thereby influencing central nervous system structures involved in stress and emotional regulation [11,12]. In addition, photobiomodulation may exert neuromodulatory and anti-inflammatory effects that could further contribute to symptom improvement in anxiety-related conditions [18–20]. LLLT-AT has also demonstrated clinical applicability, a favorable safety profile, and a non-invasive nature [12,16,17]."
• "Despite these encouraging findings, important gaps remain regarding the effectiveness of LLLT-AT for anxiety symptoms. Previous systematic reviews have evaluated the effects of conventional auriculotherapy for a range of health conditions; however, these reviews primarily examined needle-, seed-, acupressure-, or electrically based interventions rather than laser auriculotherapy [11]. More recently, LLLT-AT has shown promising results for musculoskeletal pain [17], yet its effectiveness for anxiety symptoms has not been comprehensively synthesized. In addition, preliminary evidence suggests considerable variability in laser parameters, stimulated acupoints, intervention protocols, treatment duration, and outcome measures, which may limit comparability across studies [12,17,24,25]. Therefore, this systematic review aimed to evaluate the effects of LLLT-AT on anxiety symptoms across diverse populations. The primary outcome was anxiety symptom reduction, whereas pain intensity, disability, and adverse events were evaluated as secondary outcomes."

 

Methods

(4) The eligibility criteria are generally well described. However, the inclusion of studies using LLLT-AT ‘in combination with other interventions, provided that the effects of the laser intervention could be reasonably interpreted’ is a vague formulation that introduces selection bias risk. The authors must detail how this criterion was operationalised in practice (e.g., what specific decision rule was applied to determine whether the laser’s effects were ‘reasonably interpretable’ within a combined design).

R: We thank the reviewer for these thoughtful comments, which we considered highly pertinent. In response, we revised the eligibility criteria to better define the characteristics of the included populations and to clarify the operational decision rule for studies evaluating LLLT-AT in combination with co-interventions. Specifically, we clarified that eligible studies included adults from clinical or non-clinical populations in whom anxiety symptoms were assessed using validated instruments, and that combination interventions were eligible only when identical co-interventions were applied across study groups, allowing the specific contribution of LLLT-AT to be interpreted. The revised Methods now include the following statement:

• "Eligible studies were randomized controlled trials (RCTs; parallel or crossover design) enrolling adults (≥18 years) from clinical or non-clinical populations in whom anxiety symptoms were assessed using validated anxiety assessment instruments, such as the State–Trait Anxiety Inventory (STAI), Beck Anxiety Inventory (BAI), Generalized Anxiety Disorder-7 (GAD-7), or Numeric Rating Scale for Anxiety (NRS-Anxiety). Interventions included LLLT-AT or auricular photobiomodulation therapy, administered either alone or in combination with other interventions, provided that identical co-interventions were applied across study groups and that LLLT-AT (or its placebo/sham equivalent) represented the principal between-group difference, thereby allowing the specific effect of the laser intervention to be interpreted."

 

(5) The inclusion of participants with ‘anxiety symptoms’ (without a formal diagnosis) alongside those with ‘diagnosed anxiety disorders’ generates a significant source of indirectness, which the GRADE assessment acknowledges. However, this heterogeneity is not sufficiently discussed in the Methods section. The authors should specify a priori how they planned to address this distinction in subgroup analyses, and justify why pooling these populations is methodologically appropriate.

R: Regarding the concern about indirectness, we agree that the heterogeneity of the included populations represents an important methodological consideration. Consistent with GRADE guidance, indirectness was assessed according to the applicability of the evidence with respect to the review question, considering differences in populations, interventions, comparators, and outcomes [1,2]. As can be seen in the revised GRADE evidence profiles, the indirectness domain was downgraded (rated as serious) for all critical outcomes because the available evidence was derived from markedly heterogeneous clinical populations. Consequently, the certainty of the evidence was reduced accordingly. The corresponding revisions to the GRADE evidence profiles have been highlighted in purple in the revised manuscript to facilitate their identification and review.

1. Guyatt GH, Oxman AD, Kunz R, Woodcock J, Brozek J, Helfand M, et al. GRADE guidelines: 8. Rating the quality of evidence—indirectness. J Clin Epidemiol. 2011;64(12):1303-10. doi:10.1016/j.jclinepi.2011.04.014.
2. Guyatt GH, Oxman AD, Akl EA, Kunz R, Vist G, Brozek J, et al. GRADE guidelines: 1. Introduction—GRADE evidence profiles and summary of findings tables. J Clin Epidemiol. 2011;64(4):383-94. doi:10.1016/j.jclinepi.2010.04.026.

 

(6) The search is described as last updated on May 24, 2026. An inconsistency was identified: while the abstract reports this date, the PROSPERO registration date is October 1, 2025. The authors must clarify whether the updated search is aligned with the registered protocol and report any deviations or amendments made to the original protocol.

R: We sincerely thank the reviewer for this important observation. We would like to clarify that the PROSPERO registration date (October 1, 2025) corresponds to the prospective registration of the review protocol, whereas May 24, 2026, refers to the date of the final literature search update. These represent two distinct methodological milestones and are not expected to coincide. In accordance with current methodological guidance for systematic reviews, literature searches should be updated prior to the final analyses and manuscript submission to ensure that the most recent eligible evidence is captured [3,4]. Accordingly, the updated search was conducted using the same databases, search strategy, eligibility criteria, and methodological procedures specified in the registered PROSPERO protocol. To avoid any potential misunderstanding, we also revised the corresponding sentence in the Methods section as follows:

• "This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines and was prospectively registered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD420251159376). The review protocol was registered on 1 October 2025, before study selection and data extraction commenced [26,27]."

References:

3. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021;372:n71. doi:10.1136/bmj.n71.
4. Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA, editors. Cochrane Handbook for Systematic Reviews of Interventions. Version 6.5. Cochrane; 2024. Available from: https://www.training.cochrane.org/handbook

Furthermore, to ensure that the review reflected the most up-to-date evidence available, an additional literature search update was performed on 2 July 2026 using the same databases, search strategy, eligibility criteria, and methodological procedures prespecified in the registered PROSPERO protocol. In addition, the search strategy was refined by incorporating the term "anxiety symptoms", consistent with the revised review title and objective, to maximize the sensitivity of the search while maintaining the original eligibility criteria. No additional eligible studies meeting the inclusion criteria were identified. Accordingly, the PRISMA 2020 flow diagram was updated to reflect the revised search process. The complete results of the updated search for each database are presented in the Supplementary Materials (Supplementary Table 1).

 

(7) The authors state that Fleiss’ kappa was calculated for the risk of bias assessment, but the obtained value and its interpretation are not reported anywhere in the manuscript. This statistic must be explicitly reported (kappa value and interpretation according to a conventional scale) either in the Methods or in the corresponding Results subsection.

R: We sincerely thank the reviewer for this valuable suggestion. We agree that the inter-rater agreement statistic should be explicitly reported in the manuscript. Accordingly, we have revised both the Methods and Results sections. In the Methods, we clarified that inter-rater agreement among the three independent reviewers was quantified using Fleiss' kappa statistic. In the Results, we now report the observed agreement together with its interpretation according to the Landis and Koch classification. The revised text is presented below:

• Methods: "Risk of bias assessments were independently performed by three reviewers (HDB, CCH, and NAP) using the Cochrane Risk of Bias 2 (RoB 2) tool. Inter-rater agreement was quantified using Fleiss' kappa statistic. Any discrepancies among reviewers were resolved through discussion until consensus was reached. RoB visualizations were generated using the Robvis tool [35]."
• Results: "Figure 2 presents the RoB 2 assessment of the included RCTs [34]. The risk of bias assessments were independently performed by three reviewers (HDB, CCH, and NAP), and the final judgment for each study was reached by consensus. Across the six included studies, the average Fleiss' κ prior to consensus was 0.75, indicating substantial agreement according to the Landis and Koch classification. Based on the overall RoB assessment, three studies were judged as having low RoB, one study as presenting some concerns, and two studies as having high RoB. The main methodological limitations were associated with bias in measurement of the outcome (D4) and bias in selection of the reported result (D5), whereas all studies demonstrated low RoB for deviations from intended interventions (D2) and missing outcome data (D3). Most trials also showed low RoB arising from the randomization process (D1), although one study presented some concerns regarding the randomization process. RoB plots were weighted according to each study's inverse-variance contribution to the primary meta-analysis of anxiety outcomes [33,39]."

 

To facilitate the statistical analysis, the RoB 2 judgments were transformed into ordinal numerical values as follows: 1 = Low risk, 2 = Some concerns, and 3 = High risk. This numerical coding was used exclusively for the calculation of Fleiss' κ and did not modify the original RoB 2 judgments. The corresponding coding scheme and individual reviewer ratings are also provided below for complete transparency.

 

	Fernandes et al. (2023) [24]
RoB 2 domain	Evaluator 1	Evaluator 2	Evaluator 3
D1. Bias arising from the randomization process	2	2	2
D2. Bias due to deviations from intended interventions	1	1	1
D3. Bias due to missing outcome data	1	1	1
D4. Bias in measurement of the outcome	3	3	3
D5. Bias in selection of the reported result	1	2	2
Overall risk of bias	3	3	3

 

Overall Kappa
	Kappa	Asymptotic Standard Error	Z	P Value	Lower 95% Asymptotic CI Bound	Upper 95% Asymptotic CI Bound
Overall	0,832	0,167	4,967	0,000	0,504	1,160

 

	Marques [25]
RoB 2 domain	Evaluator 1	Evaluator 2	Evaluator 3
D1. Bias arising from the randomization process	1	1	1
D2. Bias due to deviations from intended interventions	1	1	1
D3. Bias due to missing outcome data	1	1	1
D4. Bias in measurement of the outcome	2	1	2
D5. Bias in selection of the reported result	1	1	1
Overall risk of bias	1	1	1

 

Overall Kappa
	Kappa	Asymptotic Standard Error	Z	P Value	Lower 95% Asymptotic CI Bound	Upper 95% Asymptotic CI Bound
Overall	0,832	0,167	4,967	0,000	0,504	1,160

 

	Lin [46]
RoB 2 domain	Evaluator 1	Evaluator 2	Evaluator 3
D1. Bias arising from the randomization process	1	1	1
D2. Bias due to deviations from intended interventions	1	1	1
D3. Bias due to missing outcome data	2	1	1
D4. Bias in measurement of the outcome	1	2	2
D5. Bias in selection of the reported result	1	2	1
Overall risk of bias	2	3	3

 

Overall Kappa
	Kappa	Asymptotic Standard Error	Z	P Value	Lower 95% Asymptotic CI Bound	Upper 95% Asymptotic CI Bound
Overall	0,513	0,185	3,231	0,026	0,050	0,776

 

	Lemos [47]
RoB 2 domain	Evaluator 1	Evaluator 2	Evaluator 3
D1. Bias arising from the randomization process	1	1	1
D2. Bias due to deviations from intended interventions	1	1	1
D3. Bias due to missing outcome data	1	1	1
D4. Bias in measurement of the outcome	3	2	3
D5. Bias in selection of the reported result	1	1	1
Overall risk of bias	2	2	2

 

Overall Kappa
	Kappa	Asymptotic Standard Error	Z	P Value	Lower 95% Asymptotic CI Bound	Upper 95% Asymptotic CI Bound
Overall	0,775	0,182	4,263	0,000	0,419	1,131

 

	Marcondes [48]
RoB 2 domain	Evaluator 1	Evaluator 2	Evaluator 3
D1. Bias arising from the randomization process	1	1	1
D2. Bias due to deviations from intended interventions	1	2	1
D3. Bias due to missing outcome data	1	1	1
D4. Bias in measurement of the outcome	1	1	2
D5. Bias in selection of the reported result	1	1	1
Overall risk of bias	1	1	1

 

Overall Kappa
	Kappa	Asymptotic Standard Error	Z	P Value	Lower 95% Asymptotic CI Bound	Upper 95% Asymptotic CI Bound
Overall	0,910	0,0036	3,859	0,000	0,862	0,970

 

	De la Barra [49]
RoB 2 domain	Evaluator 1	Evaluator 2	Evaluator 3
D1. Bias arising from the randomization process	1	1	1
D2. Bias due to deviations from intended interventions	1	1	1
D3. Bias due to missing outcome data	1	1	1
D4. Bias in measurement of the outcome	2	1	2
D5. Bias in selection of the reported result	1	1	1
Overall risk of bias	1	1	1

 

Overall Kappa
	Kappa	Asymptotic Standard Error	Z	P Value	Lower 95% Asymptotic CI Bound	Upper 95% Asymptotic CI Bound
Overall	0,638	0,236	1,856	0,063	0,445	0,899

 

(8) A continuity correction of 0.5 was applied for studies with zero-event cells in the dichotomous adverse event analyses. Given that the majority of included studies reported zero events in both groups for several outcomes (e.g., ear pain, hyperemia), this approach may artificially inflate risk ratio estimates. The authors must explicitly justify this choice over established alternatives — such as the Sweeting continuity transformation or exact methods — and acknowledge this as a limitation of the adverse event analyses.

R: We sincerely thank the reviewer for this thoughtful and constructive comment. We agree that the management of zero-event studies should be clearly described, particularly for sparse adverse event data. Following the reviewer's suggestion, we carefully revisited both our analytical procedures and the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions. We recognized that the original wording in the Methods section could be misleading, as it did not accurately describe the analytical approach used in the final meta-analysis. Consistent with the recommendations of the Cochrane Handbook, studies reporting zero events in both groups were retained in the descriptive summary but did not contribute to the pooled RR estimates because they provide no information on the relative treatment effect. Therefore, no continuity correction was applied in the final adverse event meta-analyses. The Methods section has been revised accordingly to accurately reflect the analytical approach used. The revised text is presented below:

• "In accordance with the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions, studies reporting zero events in both groups were retained in the descriptive summary but did not contribute to the pooled RR estimates because they provide no information on the relative treatment effect [39,40]."

 

(9) The application of split-comparison procedures for the three-arm trials (Lin et al.; Marcondes et al.) is correctly justified by reference to Cochrane recommendations. However, the specific procedure used to distribute the shared control group sample between the two resulting comparisons is not described. The authors must clarify how the control group sample size was handled (e.g., halved between comparisons), in accordance with the Cochrane Handbook guidance.

R: We sincerely thank the reviewer for this valuable observation. We agree that the implementation of the split-comparison procedure for multi-arm trials should be described more explicitly to improve the transparency and reproducibility of the quantitative synthesis. Accordingly, we have revised the Methods section to clarify how shared study arms were handled in the included three-arm trials. Specifically, whenever a study arm was shared across more than one pairwise comparison, its sample size was divided between the corresponding comparisons, while the original means and standard deviations were retained for continuous outcomes, in accordance with the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions. The revised text is presented below:

• "The quantitative synthesis included studies evaluating LLLT-AT either as a standalone intervention or in combination with other therapeutic modalities. For three-arm trials, whenever a study arm was shared across more than one pairwise comparison, its sample size was divided between the corresponding pairwise comparisons, while the original means and standard deviations were retained for continuous outcomes, in accordance with the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions [39]. This approach avoided double-counting of participants while preserving all relevant comparisons."

 

(10) Table 1 contains apparent inconsistencies. First, the mean age reported for Marcondes et al. (2025) is 40.3 (16.4) years, identical to the value reported for Lin et al. (2024). Given that Marcondes et al. enrolled exclusively women with advanced breast cancer undergoing chemotherapy, this coincidence should be verified to rule out a typographical error. Second, De la Barra et al. (2026) reports 25 females and 19 males, which sums to 44, not 47 as stated as the total sample. The authors must verify and correct these figures.

R: We sincerely thank the reviewer for identifying these inconsistencies. After carefully verifying the original publications and our data extraction files, we confirmed that both issues were due to typographical errors in Table 1. The mean age reported for Marcondes et al. (2025) has been corrected according to the original publication. Likewise, the total sample size reported for De la Barra et al. (2026) has been corrected to ensure consistency with the participant characteristics presented in the table. We confirm that these corrections affect only the descriptive information presented in Table 1. The original data extraction and all quantitative analyses were performed using the correct participant numbers; therefore, these typographical corrections do not affect the meta-analytic results or the conclusions of this systematic review.

(11) The weighted risk of bias analysis is well presented. However, as noted in Comment 2.3.4, the Fleiss’ kappa value must be reported in this section. Additionally, since the first author (de la Barra Ortiz H.A.) is also the principal author of one of the included studies (reference [49]: De la Barra et al., 2026), the manuscript should explicitly state whether independent reviewers blind to authorship conducted the risk of bias assessment for this specific study, in order to ensure methodological impartiality.

R: We sincerely thank the reviewer for this important observation. We agree that transparency in the risk of bias assessment process is essential. In accordance with the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions, risk of bias assessments for all included studies, including De la Barra et al. (2026), were independently performed by three reviewers, with any disagreements resolved through discussion until consensus was reached. The Cochrane Handbook recommends the use of multiple independent reviewers and consensus procedures to enhance the reliability and objectivity of risk of bias judgments. In addition, the substantial inter-rater agreement observed in our review (mean Fleiss' κ = 0.75) further supports the consistency and reliability of the assessment process. To improve transparency, we have revised the Results section as follows:

• "Figure 2 presents the RoB 2 assessment of the included RCTs [34]. The risk of bias assessments were independently performed by three reviewers, and the final RoB judgment for each study was reached by consensus. Inter-rater agreement prior to consensus was substantial (mean Fleiss' κ = 0.75), according to the Landis and Koch classification."

(12) The substantial heterogeneity identified (I² = 78.9%) is acknowledged but not rigorously explored through pre-specified subgroup analyses or meta-regression. The subgroup analysis by comparator type (no treatment, placebo, active control) is informative but insufficient to explain the observed variability. The authors are strongly encouraged to perform and report at least one additional subgroup analysis by clinical population type (e.g., TMD vs. perioperative anxiety vs. cancer vs. university students), as clinical context is a plausible source of heterogeneity.

R: We sincerely thank the reviewer for this valuable suggestion. We agree that differences in clinical populations may represent an important source of heterogeneity in the anxiety outcome. Accordingly, we performed an additional exploratory subgroup analysis according to clinical population type, including TMD-related anxiety, parental perioperative anxiety, university student anxiety, and breast cancer-related anxiety. This analysis was performed for the primary anxiety outcome, where sufficient studies and comparisons were available. However, subgroup analyses according to clinical population type could not be reliably performed for pain intensity and disability outcomes because the limited number of available studies did not allow meaningful grouping according to different clinical populations.

The results of this additional analysis have been incorporated into the revised manuscript. A new forest plot has been added as Figure 6, presenting the subgroup analysis according to clinical population type, while the adverse event analyses have been renumbered as Figure 7. In addition, a dedicated Results subsection was included, reporting the subgroup effect estimates, heterogeneity statistics, and test for subgroup differences. The corresponding certainty of evidence assessment has also been reported in the GRADE summary of findings (Table 3).

• New subsection: “Anxiety outcome (subgroup analysis according to clinical population type). Figure 6 presents the exploratory subgroup analysis according to clinical population type for post-treatment anxiety outcomes. This analysis included six studies comprising eight comparisons and was performed to investigate potential sources of the substantial heterogeneity observed in the primary anxiety analysis (I² = 78.9%). For the three-arm trials conducted by Lin et al. [46] and Marcondes et al. [48], split-comparison procedures were applied according to Cochrane recommendations by dividing the shared study arm between comparisons to avoid double-counting participants. Significant subgroup differences were identified (χ² = 31.03, df = 3, p < 0.001). LLLT-AT showed significant effects in participants with TMD-related anxiety (SMD = −1.10; 95% CI: −1.51 to −0.68; I² = 0%) and parental perioperative anxiety (SMD = −1.45; 95% CI: −2.18 to −0.71; I² = 0%). Conversely, no significant effects were observed for university student anxiety (SMD = 0.07; 95% CI: −0.31 to 0.45) or breast cancer-related anxiety (SMD = 0.10; 95% CI: −0.28 to 0.47). Based on the predefined Hedges’ g thresholds, the effects observed for TMD-related anxiety and parental perioperative anxiety were classified as large, whereas those observed for university student anxiety and breast cancer-related anxiety were classified as negligible [43]”.

“According to the GRADE assessment, the certainty of evidence was low for all subgroup analyses of anxiety outcomes, including TMD-related anxiety, parental perioperative anxiety, university student anxiety, and breast cancer-related anxiety (Table 3)”.

 

(13) The sensitivity analysis excluding studies with high risk of bias (Fernandes et al. and Lin et al.) yielded a non-significant pooled effect (SMD = −0.35; 95% CI: −0.94 to 0.25; p = 0.2524). This is the most methodologically robust estimate in the entire manuscript and should receive substantially greater prominence in the Results, Discussion, and Conclusions sections. In its current form, the manuscript prioritises the overall effect estimate (which includes high-risk-of-bias studies) over the sensitivity result, which may lead readers to overestimate the evidence for LLLT-AT’s anxiolytic effects. This must be addressed and rebalanced throughout the manuscript.

R: We sincerely thank the reviewer for this important observation. We agree that the sensitivity analysis excluding studies with high RoB provides important information regarding the robustness of the estimated effect of LLLT-AT on anxiety outcomes. Although this analysis was already reported in the previous version of the manuscript, we agree that it required greater prominence and a more balanced interpretation throughout the manuscript.

Accordingly, we have revised the Abstract, Results, Discussion, Limitations, and Conclusions sections to emphasize that the exclusion of studies with high RoB resulted in an attenuated and non-significant effect estimate (SMD = −0.35; 95% CI: −0.94 to 0.25; p = 0.2524). The specific modifications are presented below:

• Abstract: "However, sensitivity analyses excluding studies with high risk of bias yielded a non-significant effect for anxiety outcomes (SMD = −0.35; 95% CI: −0.94 to 0.25). Interpretation of the findings should be considered cautiously due to substantial heterogeneity and the very low-to-low certainty of evidence for anxiety outcomes."
• Results: "The pooled analysis demonstrated no statistically significant difference between LLLT-AT and control interventions (SMD = −0.35; 95% CI: −0.94 to 0.25; p = 0.2524). Compared with the primary analysis, the exclusion of studies with high RoB resulted in a reduced effect estimate, indicating that the overall anxiety findings should be interpreted with caution."
• Discussion: "Overall, pooled analyses demonstrated reductions in post-treatment anxiety symptoms favoring LLLT-AT compared with control interventions; however, sensitivity analyses excluding studies with high RoB resulted in an attenuated and non-significant effect estimate, indicating limited robustness of the primary finding. In addition, the substantial heterogeneity and very low-to-low certainty of evidence warrant cautious interpretation."
• Limitations: "Sensitivity analysis excluding studies with high RoB resulted in an attenuated and non-significant effect estimate for anxiety outcomes, indicating that the magnitude of the observed anxiolytic effect should be interpreted cautiously."
• Conclusions: "However, sensitivity analyses excluding studies with high RoB resulted in an attenuated and non-significant effect estimate, indicating that the magnitude and robustness of this effect should be interpreted cautiously."

(14) These revisions provide a more balanced interpretation of the primary findings and the sensitivity analysis, avoiding overestimation of the certainty and magnitude of the anxiolytic effects of LLLT-AT. The adverse event meta-analyses are almost entirely driven by a single study (Lemos et al., 2024), as most included studies reported zero events in both groups. While the authors briefly acknowledge this in the text, they must be more explicit in stating that these pooled analyses are, in practice, single-study estimates artificially amplified by the continuity correction applied to zero cells. It is recommended that these results be presented descriptively or narratively rather than as formal meta-analyses, and that the pooled risk ratio estimates be interpreted with the utmost caution.

R: We sincerely thank the reviewer for this valuable comment. We agree that adverse event analyses require a cautious interpretation due to the limited number of studies reporting events. As clarified in our previous response regarding zero-event studies, the statement about applying a continuity correction of 0.5 reflected an initially planned approach but did not accurately represent the final analytical procedure used. No continuity correction was applied in the final adverse event analyses; therefore, the pooled RR estimates were not artificially amplified by adding values to zero-event cells.

In accordance with the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions, studies reporting zero events in both groups were retained descriptively but did not contribute to pooled RR estimates, as these studies provide no information regarding the relative treatment effect.

Nevertheless, we agree with the reviewer that most adverse event estimates were based on a limited number of informative comparisons, with a substantial contribution from Lemos et al. (2024). Therefore, we have revised the Results section to explicitly emphasize this limitation and to ensure a more cautious interpretation of the adverse event findings. The revised text is presented below:

• "Adverse effects. Figures 7A–7D present the analyses of adverse effects associated with LLLT-AT compared with control interventions [24,25,46–49]. Reported adverse effects included ear pain, ear hyperemia, itchy ear, and intervention-related headache. Dichotomous outcomes were analyzed using Mantel–Haenszel RRs with 95% confidence intervals. Studies reporting zero events in both groups were retained descriptively and did not contribute to pooled RR estimates, according to Cochrane recommendations [40]. Most adverse-effect estimates were based on a limited number of informative comparisons, with a substantial contribution from Lemos et al. [47], and should therefore be interpreted cautiously."

(15) Table 3 contains an inconsistency in the sample sizes for the outcome ‘Disability (JFLS-8): LLLT-AT vs. Placebo’: the table reports 44 participants in the LLLT-AT group and 44 in the control group (total = 88), whereas the text reports 31 in the LLLT-AT group and 26 in the control group (total = 57). The authors must verify and correct these values to ensure internal consistency between tables and text.

R: We sincerely thank the reviewer for carefully identifying this inconsistency. After verifying the data extraction files and quantitative analyses, we confirmed that this discrepancy was due to a typographical error during the preparation of Table 3, where values from another cell were inadvertently copied. The sample sizes for the disability outcome (JFLS-8: LLLT-AT vs. placebo) have now been corrected in Table 3 to ensure consistency with the values reported in the text. We confirm that this correction only affects the descriptive information presented in the table and does not affect the pooled effect estimates, statistical analyses, or conclusions of the review.

 

Discussion

(16) As indicated in Comment 2.4.4, the manuscript in its current form does not adequately balance the presentation of the primary pooled effect (significant) against the sensitivity analysis (non-significant). The discussion and conclusions must reflect that the evidence is, at best, preliminary and not conclusive for the anxiety outcome, given the fragility of the effect when high-risk-of-bias studies are excluded.

R: We sincerely thank the reviewer for this important observation. We agree that the Discussion and Conclusions should clearly reflect the preliminary and non-conclusive nature of the current evidence for anxiety outcomes, particularly given the attenuation and loss of statistical significance observed in the sensitivity analysis excluding studies with high RoB.

Following this comment, and in line with the revisions made in response to the previous methodological comments, we have further revised the Discussion to provide a more cautious and balanced interpretation of the evidence. Specifically, we clarified that the proposed mechanisms remain hypothetical, that the pooled anxiety estimate should be interpreted as an exploratory synthesis across heterogeneous clinical contexts, and that the clinical and public health implications should be considered hypothesis-generating rather than direct recommendations. The following sentences were incorporated into the revised Discussion:

• “The potential anxiolytic effects of LLLT-AT remain insufficiently established at the mechanistic level. Current explanations are largely based on indirect evidence from auricular stimulation, vagal modulation, needle-based auriculotherapy, and broader photobiomodulation research rather than on direct mechanistic studies of LLLT-AT. Therefore, the mechanisms discussed below should be interpreted as plausible hypotheses rather than confirmed pathways.”
• “Although anxiety symptoms constituted the common outcome across studies, the included populations differed substantially in clinical context, psychosocial burden, comorbidity profile, and intervention setting. Therefore, the pooled anxiety estimate should be interpreted as an exploratory synthesis across heterogeneous anxiety-related contexts rather than as evidence for a uniform effect of LLLT-AT across all populations.”
• “Given the limited number of trials, substantial heterogeneity, and very low-to-low certainty of evidence for anxiety outcomes, the clinical and public health implications of these findings should be considered hypothesis-generating and should not be interpreted as direct clinical or public health recommendations.”

In addition, as described in our response to the previous comment, we also revised the Abstract, Results, Limitations, and Conclusions sections to emphasize that the sensitivity analysis excluding studies with high RoB yielded an attenuated and non-significant effect estimate. Therefore, the revised manuscript now consistently presents the evidence for anxiety outcomes as preliminary, exploratory, and requiring cautious interpretation rather than as conclusive evidence of efficacy.

 

(17) The neurobiological mechanisms section is well documented but contains speculative claims that are not directly supported by LLLT-AT-specific evidence. Much of the mechanistic reasoning is inferred by analogy from transcranial photobiomodulation, non-auricular vagal stimulation, or needle-based auriculotherapy studies. The authors must clearly distinguish between mechanisms with direct LLLT-AT evidence and those inferred by analogy, using appropriately cautious language (‘may’, ‘has been proposed’, ‘remains to be established’).

R: Specifically, we clarified that direct evidence involving LLLT-AT is currently limited mainly to clinical outcomes, treatment acceptability, safety, and intervention parameters reported in randomized controlled trials, whereas the underlying neurobiological mechanisms remain largely inferential. We also revised the wording throughout the section to use more cautious language, including “may,” “has been proposed,” “potential,” “plausible,” “compatible with the hypothesis,” and “remain incompletely understood.”

The revised section now begins with the following clarification:

• “The potential anxiolytic effects of LLLT-AT remain insufficiently established at the mechanistic level. Current explanations are largely based on indirect evidence from auricular stimulation, vagal modulation, needle-based auriculotherapy, and broader photobiomodulation research rather than on direct mechanistic studies of LLLT-AT. Therefore, the mechanisms discussed below should be interpreted as plausible hypotheses rather than confirmed pathways. Importantly, direct evidence involving LLLT-AT is currently limited mainly to clinical outcomes, treatment acceptability, safety, and intervention parameters reported in randomized controlled trials [24,25,46–49], whereas the underlying neurobiological mechanisms remain largely inferential and require direct experimental confirmation.”

In addition, causal or definitive wording was replaced by more cautious phrasing. For example, the sentence describing the overall mechanistic rationale was revised as follows:

• “From a theoretical perspective, these potential effects may be partially explained by neurophysiological mechanisms associated with both auriculotherapy and photobiomodulation.”

 

Similarly, the interpretation of the broader mechanistic evidence was softened to avoid overstating the findings:

• “Although the precise neurophysiological pathways underlying these effects remain incompletely understood, the current findings are compatible with the hypothesis that LLLT-AT may exert multidimensional effects involving psychological, autonomic, and neurophysiological regulation.”

These revisions explicitly frame the mechanistic discussion as biologically plausible but not definitively established, and they distinguish the direct clinical evidence for LLLT-AT from mechanistic explanations inferred by analogy from auricular stimulation, vagal modulation, needle-based auriculotherapy, and broader photobiomodulation research.

 

(18) The discussion on clinical heterogeneity is appropriate but should more explicitly address the implications of combining populations as diverse as TMD patients, parents in a surgical waiting room, university students, and women with advanced breast cancer in a single meta-analysis. The authors should provide a more critical reflection on whether the shared denominator of ‘anxiety symptoms’ is sufficient to justify quantitative synthesis across such markedly different clinical contexts, psychosocial burdens, and comorbidity profiles.

R: We agree that the clinical and methodological heterogeneity of the included studies required a more explicit and cautious discussion. Although anxiety symptoms were the common outcome across studies, the included populations represented markedly different clinical contexts, which limits the interpretation of the pooled estimate as a uniform treatment effect.

Accordingly, we revised the Discussion by adding a dedicated subsection on Clinical heterogeneity and protocol variability. In this section, we clarified that the pooled anxiety estimate should be interpreted as an exploratory synthesis across heterogeneous anxiety-related contexts rather than as conclusive evidence of a consistent effect of LLLT-AT across all populations. The revised manuscript now states:

• “Although anxiety symptoms constituted the common outcome across studies, the included populations differed substantially in clinical context, psychosocial burden, comorbidity profile, and intervention setting. Therefore, the pooled anxiety estimate should be interpreted as an exploratory synthesis across heterogeneous anxiety-related contexts rather than as evidence for a uniform effect of LLLT-AT across all populations.”

We also expanded the discussion of potential sources of heterogeneity, including differences in clinical populations, psychosocial burden, comorbidities, laser dosimetry, number of auricular points, treatment duration, anxiety assessment instruments, comparator interventions, and concomitant therapies. These issues were explicitly linked to the indirectness concerns considered in the GRADE assessment.

In addition, the clinical and public health implications were toned down to avoid overinterpretation of the findings. The revised Discussion now states:

• “Given the limited number of trials, substantial heterogeneity, and very low-to-low certainty of evidence for anxiety outcomes, the clinical and public health implications of these findings should be considered hypothesis-generating and should not be interpreted as direct clinical or public health recommendations.”

Finally, this revision was aligned with changes made in other sections of the manuscript, including the Abstract, Results, Limitations, and Conclusions, where we now emphasize that the evidence for anxiety outcomes remains preliminary, exploratory, and uncertain, particularly considering the non-significant sensitivity analysis after excluding studies with high risk of bias.

 

(19) The dosimetric considerations section is informative but employs a prescriptive, forward-looking tone that the current evidence cannot support. The parameter ranges described (wavelength 660–905 nm; energy density 0.19–4 J/cm²; treatment time 20–56 seconds per point) are so broad as to constitute a description of what has been used rather than evidence-based optimal dosimetry. The authors must use more cautious language and explicitly acknowledge that these ranges reflect the existing literature and do not constitute dosimetric recommendations.

R:  We agree with the reviewer’s comment. The section “4.4. Dosimetric and Protocol Considerations” has been revised to ensure that the reported dosimetric parameters are presented as a descriptive summary of the protocols used in the included studies, rather than as evidence-based recommendations.

This section was rewritten in response to the reviewer’s suggestions and also to maintain consistency with the related concerns raised by Reviewer 2 regarding the preliminary nature of the evidence, clinical heterogeneity, and the need to avoid prescriptive interpretations. Specifically, we clarified that the current evidence remains insufficient to establish definitive dosimetric recommendations for LLLT-AT in anxiety-related conditions. We also added the following sentence to explicitly address the reviewer’s concern:

• “These ranges should be interpreted as a descriptive summary of the dosimetric approaches reported in the existing literature, rather than as evidence-based recommendations for optimal LLLT-AT dosing.”

In addition, we softened the forward-looking interpretation by replacing the expression “superior clinical outcomes” with “differences in clinical outcomes”, to avoid implying that any specific wavelength, energy density, irradiation time, or number of auricular points can currently be recommended as optimal. The revised section now states:

• “Current evidence remains insufficient to establish definitive dosimetric recommendations for LLLT-AT in anxiety-related conditions. Nevertheless, the protocols identified in this systematic review provide a preliminary overview of the dosimetric parameters currently investigated. Across the included studies, wavelengths ranged from 660 to 905 nm, energy density from 0.19 to 4 J/cm², the number of irradiated auricular points from 4 to 16, and irradiation time from 20 to 56 seconds per point [24,25,46–49]. These ranges should be interpreted as a descriptive summary of the dosimetric approaches reported in the existing literature, rather than as evidence-based recommendations for optimal LLLT-AT dosing. Future randomized controlled trials should determine whether specific dosimetric parameters are associated with differences in clinical outcomes, while prioritizing standardized photobiomodulation reporting, dose–response analyses, protocol harmonization, and longer-term follow-up assessments.”

 

(20) The first and corresponding author (de la Barra Ortiz H.A.) is also the lead author of one of the six included studies (reference [49]: De la Barra et al., 2026, Int. J. Environ. Res. Public Health), which is the study with the highest PEDro score (9/10) and contributes substantially to the anxiety and disability analyses. Although this does not invalidate the review, the manuscript must explicitly address this potential conflict of interest in the Limitations section and declare whether specific methodological safeguards were implemented (e.g., independent external reviewers conducting the risk of bias assessment for study [49]).

R: We agree that the authorial overlap between the first and corresponding author of this systematic review and one of the included RCTs [49] should be explicitly acknowledged. This issue has now been addressed in the Limitations section. We clarified that study [49] was included because it met the prespecified eligibility criteria and was handled using the same methodological procedures applied to all included studies. To reduce potential bias, data extraction, PEDro methodological quality assessment, and RoB judgments for this study were independently verified by other reviewers, and any discrepancies were resolved by consensus. We also clarify that the PEDro score was used descriptively to summarize methodological quality and was not used to determine study inclusion or statistical weighting in the meta-analysis. The following text was added to the Limitations section:

• “An additional consideration is that the first and corresponding author of this systematic review was also the lead author of one of the included RCTs [49]. This authorial overlap was addressed by applying the same prespecified eligibility, data extraction, PEDro methodological quality assessment, and RoB assessment procedures used for all included studies, with data extraction, PEDro scoring, and RoB judgments reviewed by the other reviewers and resolved by consensus.”

This revision explicitly acknowledges the authorial overlap while clarifying the methodological safeguards used to minimize bias in the inclusion, extraction, PEDro assessment, and RoB appraisal of study [49].

 

Conclusions

(21) The current conclusions state that ‘LLLT-AT was associated with reduced anxiety symptoms across different clinical populations’ without adequately qualifying that this effect did not hold in the most methodologically rigorous sensitivity analysis. The conclusions must be reformulated to accurately reflect the state of the evidence: the effect is preliminary, not robust to sensitivity analyses, and based on a small body of highly heterogeneous studies. The tone of the conclusions must be consistent with the very low to low GRADE certainty rating assigned to the primary outcome.

R: We agree with the reviewer’s comment that the Conclusions section should more accurately reflect the uncertainty of the current evidence, particularly considering the results of the sensitivity analysis, the substantial heterogeneity, and the very low-to-low certainty of evidence according to the GRADE assessment. Accordingly, the Conclusions section has been reformulated to avoid presenting the pooled effect as definitive evidence of efficacy. We now explicitly state that the observed effects should be considered preliminary and acknowledge that the anxiety outcome was not robust after excluding studies with high risk of bias. The revised Conclusions section now states:

“The current evidence suggests that LLLT-AT may be associated with reductions in anxiety symptoms across different clinical populations; however, these findings should be considered preliminary due to the limited number of available RCTs, substantial clinical and methodological heterogeneity, and very low certainty of evidence for anxiety outcomes. Importantly, sensitivity analyses excluding studies with high risk of bias showed that the pooled effect for anxiety was not statistically significant, indicating that the robustness of the current evidence remains limited.”

In addition, the final interpretation was modified to emphasize the need for further confirmation:

• “Future high-quality RCTs with rigorous methodological designs, standardized intervention protocols, and longer follow-up assessments are warranted to confirm these preliminary findings and reduce the remaining uncertainty regarding the effectiveness of LLLT-AT for anxiety-related symptoms.”

 

(22) The clinical and public health implications presented in Section 4.6 and summarised in the conclusions exceed what the available evidence can support. Statements suggesting the potential integration of LLLT-AT into primary care or community-based settings should be clearly framed within the context of the available evidence quality and must not be presented as direct clinical recommendations.

R: We agree with the reviewer that the clinical and public health implications should be interpreted according to the quality and certainty of the available evidence, and that the current findings should not be presented as direct recommendations for implementation in clinical or community-based settings. Accordingly, we revised the Public Health Implications section to provide a more cautious interpretation and to clearly differentiate potential future applications from current evidence-based recommendations. Specifically, statements regarding the integration of LLLT-AT into healthcare settings were reformulated to emphasize that further research is required before implementation can be considered. The revised manuscript now states:

• “These characteristics support further investigation of auriculotherapy as a potential complementary strategy within multidisciplinary healthcare contexts, particularly when safe, non-invasive, and acceptable therapeutic options are being explored.”

In addition, we added an explicit statement reflecting the limitations of the current evidence:

• “However, considering the limited number of available trials, substantial heterogeneity, and very low-to-low certainty of evidence, the potential public health implications of LLLT-AT remain preliminary and should not be interpreted as recommendations for implementation into routine healthcare settings. Further effectiveness and implementation research is required before its role in healthcare systems can be established.”

 

Errors

(23) The intervention column header in Table 3 uses ‘LLL-AT’ instead of ‘LLLT-AT’. This typographical error must be corrected throughout the table.

R: We thank the reviewer for identifying this typographical error. The abbreviation “LLL-AT” in the intervention column header of Table 3 has been corrected to “LLLT-AT” throughout the table.

 

(24) Throughout the manuscript, inconsistencies in reference numbering were identified. For example, in Section 3.3, the citation ‘(24–29)’ implies a consecutive range, whereas the six included studies correspond to references [24], [25], [46], [47], [48], and [49] — not a consecutive sequence. This pattern is repeated at several other points in the text. The authors must conduct a thorough review of all in-text citations to ensure consistency with the reference list.

R: We conducted a full review of the in-text citations and corrected the inconsistent citation formatting throughout the manuscript. In particular, the erroneous citation range “(24–29)” in Section 3.3 was corrected to “[24,25,46–49]”, and other instances using parentheses instead of square brackets were also corrected to ensure consistency with the reference list and journal style.

References [7] (Wehry et al., 2015) and [8] (Creswell et al., 2014) address the assessment and management of anxiety disorders in children and adolescents, not in adults. Their use to support standard treatment approaches for adult anxiety disorders is potentially misleading. The authors should replace these with references specifically addressing adult populations.

R: We sincerely thank the reviewer for this observation. We agree that the previously cited references primarily addressed anxiety assessment and management in children and adolescents and were therefore not the most appropriate sources to support treatment strategies in adult populations. Accordingly, these references have been replaced with references specifically focused on adult anxiety disorders and their management, including pharmacological and non-pharmacological approaches.

Replaced references:

• Penninx BWJH, Pine DS, Holmes EA, Reif A. Anxiety disorders. The Lancet. 2021;397(10277):914–927. doi:10.1016/S0140-6736(21)00359-7
• Bandelow B, Michaelis S, Wedekind D. Treatment of anxiety disorders. Dialogues in Clinical Neuroscience. 2017;19(2):93–107. doi:10.31887/DCNS.2017.19.2/bbandelow

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We thank the reviewer once again for the valuable and constructive feedback provided during this review process. We believe that the revisions undertaken in response to these comments have substantially strengthened the manuscript by improving methodological transparency, reporting accuracy, and the balance in the interpretation of the available evidence. We appreciate the reviewer’s contribution to enhancing the overall rigor and clarity of our work.

 

Thank you for your time and consideration.

Sincere regards,

The research team

 

Reviewer 3 Report

Comments and Suggestions for Authors

The strength of this review: Table and figure of laser parameters and auricular acupoint maps of included studies are useful to the clinical application.

The weakness of this review

1. Sensitivity analysis of post-treatment anxiety symptoms demonstrated no statistically significant difference between the groups. It is difficult to confirm the effect of LLLT-AT on anxiety. It is better to reveal the authors' discussions more.

2. Laser therapy can have a bad effect on the eyes. When laser therapy is performed on the ear located next to the eyes, measures to minimize the effect on the eyes are needed. Referencing this in the article can help clinical use.

3. In the discussion, the contents of the mechanisms of LLLT-AT are too long. This review is to verify the effect size, safety, and evidence level rather than to review the mechanism. There is a brief description of the mechanism of LLLT-AT in the introduction, so it is better to delete it in the discussion. 

Author Response

III. REVIEWER 3 COMMENTS AND SUGGESTIONS

Comments to the author (if any):

The strength of this review: Table and figure of laser parameters and auricular acupoint maps of included studies are useful to the clinical application

 

Dear Reviewer 3,

We thank you for your careful review of our manuscript and for your positive comments regarding the usefulness of the laser parameter table and auricular acupoint figure for clinical interpretation. We greatly appreciate your constructive suggestions, which helped us improve the balance of the Discussion, strengthen safety considerations, and provide a more cautious interpretation of the available evidence. We have carefully addressed each of your comments, and the corresponding revisions are detailed below.

(1) Sensitivity analysis of post-treatment anxiety symptoms demonstrated no statistically significant difference between the groups. It is difficult to confirm the effect of LLLT-AT on anxiety. It is better to reveal the authors' discussions more.

R: We thank the reviewer for this important comment. We agree that the non-significant sensitivity analysis after excluding studies with high risk of bias reduces the certainty and robustness of the observed effect of LLLT-AT on anxiety outcomes. Therefore, we revised the manuscript to avoid overinterpretation of the pooled findings and to provide a more balanced discussion of the current evidence. Specifically, the Abstract, Discussion, Limitations, and Conclusions sections were revised to emphasize that the observed anxiolytic effect should be considered preliminary rather than confirmatory, considering the sensitivity analysis results, substantial heterogeneity, and the very low-to-low certainty of evidence according to GRADE. The Discussion section has been expanded to explicitly address this issue:

• “Overall, pooled analyses demonstrated reductions in post-treatment anxiety symptoms favoring LLLT-AT compared with control interventions; however, sensitivity analyses excluding studies with high RoB resulted in an attenuated and non-significant effect estimate, indicating that the observed anxiolytic effect is sensitive to methodological quality and should therefore be considered preliminary rather than confirmatory. In addition, substantial heterogeneity and very low-to-low certainty of evidence warrant cautious interpretation.”

Furthermore, the Limitations section was revised to reinforce this interpretation:

• “Sensitivity analysis excluding studies with high RoB resulted in an attenuated and non-significant effect estimate for anxiety outcomes, indicating that the magnitude of the observed anxiolytic effect should be interpreted cautiously.”

Finally, the Conclusions were reformulated to reflect the preliminary nature of the evidence and the need for further confirmation through high-quality RCTs.

 

(2) Laser therapy can have a bad effect on the eyes. When laser therapy is performed on the ear located next to the eyes, measures to minimize the effect on the eyes are needed. Referencing this in the article can help clinical use.

R: We thank the reviewer for this valuable comment. We agree that, although LLLT-AT is considered a non-invasive intervention and no ocular adverse events were reported in the included studies, appropriate laser safety considerations are important due to the anatomical proximity of auricular points to the eyes.

Accordingly, we have revised the Adverse Effects and Safety Profile section to include a specific statement regarding preventive ocular safety measures during LLLT-AT application. The following sentence has been added:

• “In addition, although no ocular adverse events were reported, appropriate laser safety measures, including avoidance of direct ocular exposure and use of eye protection when required, should be considered due to the anatomical proximity of auricular points to the eyes.”

 

(3) In the discussion, the contents of the mechanisms of LLLT-AT are too long. This review is to verify the effect size, safety, and evidence level rather than to review the mechanism. There is a brief description of the mechanism of LLLT-AT in the introduction, so it is better to delete it in the discussion.

R: We thank the reviewer for this valuable comment. We agree that the primary objective of this systematic review and meta-analysis is to evaluate the clinical effectiveness, safety profile, and certainty of evidence of LLLT-AT rather than to provide an extensive discussion of its mechanisms.

Following the reviewer’s suggestion, we substantially shortened the Neurophysiological Mechanisms of LLLT-AT section. Redundant explanations regarding autonomic pathways, central nervous system structures, photobiomodulation mechanisms, auricular acupoints, and pain modulation were removed where appropriate or condensed to maintain the focus of the Discussion on the clinical findings.

Considering the overall recommendations received during the review process, a concise mechanistic framework was retained to clarify the biological rationale of LLLT-AT while avoiding overinterpretation. These revisions reduced the length of the mechanistic discussion and ensured that the manuscript remains focused on effect estimates, safety outcomes, and certainty of evidence.

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We thank the reviewer once again for the constructive comments. We believe that the revisions performed have improved the clarity, focus, and overall interpretation of the manuscript.

 

Thank you for your time and consideration.

Sincere regards,

The research team

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors The authors received the necessary justifications and adjustments.