Autism spectrum disorder (ASD) is classified by the World Health Organization in ICD-11 (International Classification of Diseases, 11th Revision; code: 6A02) under neurodevelopmental disorders and is defined by persistent deficits in initiating and sustaining reciprocal social interaction and social communication and by a range of restricted, repetitive, and inflexible patterns of behavior, interests, or activities [1]. The DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition), as published by the American Psychiatric Association, similarly conceptualizes ASD, with the diagnostic criteria largely consistent with ICD-11 [2]. ASD presents with wide variability in symptom severity and functional impairment, necessitating individualized therapeutic approaches.

Recent epidemiological data indicate a continuing rise in ASD prevalence worldwide. According to the Centers for Disease Control and Prevention, approximately 1 in 31 children in the United States has been diagnosed with ASD [3]. Similar trends have been reported globally, including in Asian countries. In India, increasing awareness, improved diagnostic services, and expanded epidemiological surveillance have contributed to greater recognition of ASD. However, despite this increasing disease burden, access to comprehensive, evidence-based interventions remains limited, and universally accepted treatment guidelines addressing the multidimensional nature of ASD are lacking.

Although the precise etiology of ASD remains incompletely understood, current evidence suggests that its pathogenesis is multifactorial, involving complex interactions among genetic susceptibility, environmental influences, immune dysregulation, oxidative stress, neuroinflammation, and abnormalities of the gut-brain axis [4-7]. Increasing evidence indicates that persistent neuroinflammation and altered immune responses contribute to behavioral and cognitive dysfunction in ASD. Elevated circulating inflammatory mediators, including high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), have been consistently reported in individuals with ASD, supporting the hypothesis that inflammatory pathways may influence disease severity and therapeutic response [7]. Although these biomarkers are not diagnostic of ASD, they provide valuable insight into underlying biological mechanisms and may serve as exploratory indicators of treatment response.

Current management of ASD primarily relies on multidisciplinary nonpharmacological interventions, including behavioral, speech-language, occupational, and educational therapies. These interventions improve functional outcomes but often require prolonged implementation and variable caregiver engagement. Pharmacological therapies are generally reserved for associated symptoms such as irritability, aggression, hyperactivity, or sleep disturbances and have limited efficacy in addressing the core deficits of ASD. Furthermore, emerging pharmacological agents, including intranasal oxytocin and N-acetylcysteine, remain investigational, with inconsistent evidence supporting routine clinical use [8]. Consequently, there is growing interest in adjunctive interventions capable of complementing conventional therapy through multiple biological and behavioral mechanisms.

Ayurveda provides a holistic and individualized approach to health that emphasizes restoration of physiological balance through multimodal therapeutic strategies. Although ASD is not explicitly described in classical Ayurvedic literature, many of its clinical manifestations resemble those described under Unmada, a condition characterized by disturbances in cognition, behavior, speech, and emotional regulation [9]. Among the Ayurvedic interventions traditionally indicated for cognitive disorders, Saraswata Ghrita and the polyherbal formulation Ayush Manas have received increasing attention because of their reported neuroprotective, antioxidant, anti-inflammatory, and cognition-enhancing properties. Their constituent herbs, including Brahmi (Bacopa monnieri), Ashwagandha (Withania somnifera), Jyotishmati (Celastrus paniculatus), and Mandukaparni (Centella asiatica), have demonstrated beneficial effects on neuronal function, oxidative stress, neuroinflammation, and synaptic plasticity in experimental and early clinical studies [10-14]. Therefore, inflammatory biomarkers, including hs-CRP, IL-6, and TNF-α, have been incorporated as exploratory mechanistic outcome measures to evaluate whether adjunctive Ayurvedic therapy is associated with modulation of inflammatory processes alongside improvements in behavioral and functional outcomes. Collectively, these findings provide a biologically plausible rationale for evaluating these formulations as adjuncts to conventional ASD therapy [15,16].

As ASD affects multiple domains of functioning, assessment of treatment response requires validated multidimensional outcome measures. Accordingly, this study employs the Autism Treatment Evaluation Checklist (ATEC) as the primary outcome measure and incorporates secondary outcome measures, including the Indian Scale for Assessment of Autism (ISAA) to measure severity across core behavioral domains, the Wechsler Intelligence Scale for Children (WISC) for intellectual ability, the Vineland Adaptive Behavior Scale (VABS), particularly the Daily Living Skills, to determine functional independence, and Quality of Life in Autism Questionnaire (QoLA) to evaluate parental quality of life, and exploratory inflammatory biomarkers. Together, these measures enable comprehensive evaluation of both clinical and potential biological effects of the intervention [17-20].

Despite encouraging preclinical evidence and limited clinical observations supporting Ayurvedic interventions in neurodevelopmental disorders, robust randomized controlled trials (RCTs) evaluating their adjunctive role in ASD remain scarce. High-quality evidence is therefore required to determine whether integration of Ayurveda with conventional therapy can improve behavioral, cognitive, adaptive, and family-centered outcomes while also providing insight into potential biological mechanisms.

The study describes the protocol for a randomized, open-label, active-controlled clinical trial evaluating a comprehensive Ayurveda intervention comprising Saraswata Ghrita and Ayush Manas as an adjunct to conventional therapy in children with ASD. The trial aims to determine whether this integrative approach improves behavioral symptoms, intellectual functioning, adaptive behavior, and parental quality of life while exploring changes in inflammatory biomarkers, including hs-CRP, IL-6, and TNF-α. The findings are expected to strengthen the evidence base for integrative ASD management and inform the development of standardized multidisciplinary treatment strategies.

The primary objective of this study is to evaluate the efficacy of the comprehensive Ayurveda approach as an add-on to the conventional therapy in children with ASD. Additionally, the study aims to assess the intellectual ability and daily living adaptive functioning of the children, as well as the quality of life of their parents.

This study is a randomized, open-label, parallel-group, active-controlled clinical trial with a 1:1 allocation ratio, designed in accordance with the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) 2025 guidelines (Checklist 1). The trial evaluates the efficacy of a comprehensive Ayurveda intervention comprising Saraswata Ghrita and Ayush Manas as an adjunct to conventional therapy in children with ASD. Participants will be allocated to either conventional therapy alone (control group) or conventional therapy combined with the Ayurvedic intervention (intervention group) for 6 months. The schedule of enrollment, interventions, and outcome assessments is presented in Table 1.

The trial is being conducted at the Central Ayurveda Research Institute (CARI), Bengaluru, India, in collaboration with selected autism therapy centers that provide standard multidisciplinary ASD care. Conventional interventions, including behavioral, speech-language, occupational, and special education therapies, are delivered at the collaborating centers, whereas study enrollment, clinical assessments, laboratory investigations, dispensing of study medications, and follow-up evaluations are undertaken at CARI.

The study is conducted by a multidisciplinary research team comprising the principal investigator, coinvestigators, Ayurveda physicians, psychologists, pediatricians, laboratory personnel, and trained study coordinators. The principal investigator is responsible for overall trial conduct, protocol compliance, participant safety, and data integrity. Coinvestigators oversee clinical assessments and outcome evaluations, while trained study personnel are responsible for participant recruitment, data collection, documentation, follow-up, and maintenance of study records in accordance with Good Clinical Practice (GCP) and institutional standard operating procedures.

Children diagnosed with ASD and receiving conventional therapy at participating autism therapy centers are screened for eligibility before enrollment. Recruitment commenced in December 2025 and will continue until the required sample size is achieved. Written informed consent is obtained from parents or legal guardians before any study-related procedures are performed.

The eligibility criteria were developed to recruit a clinically homogeneous population while ensuring participant safety and minimizing potential confounding factors that could influence behavioral, cognitive, adaptive, or inflammatory outcomes (Textbox 1). The selected age range (6‐15 y) corresponds to the developmental period during which the standardized assessment instruments used in this study are valid and behavioral interventions can be implemented consistently.

Short-term medications prescribed for acute illnesses that are not expected to influence study outcomes will not constitute grounds for exclusion and will be documented as concomitant medications.

The selected eligibility criteria are intended to minimize clinical heterogeneity while ensuring that changes observed during follow-up can be more reliably attributed to the study intervention rather than to major comorbid conditions or concurrent pharmacological therapies.

To ensure consistent implementation of the study protocol, all investigators, study coordinators, and collaborating care providers will receive standardized training before participant enrollment. Periodic monitoring visits, supervisory meetings, and protocol audits will be conducted throughout the trial to identify and address protocol deviations.

Participant adherence will be evaluated using multiple complementary measures. Caregivers will receive verbal and written instructions regarding medication administration and continuation of conventional therapy. Compliance with the investigational products will be assessed through caregiver-maintained medication diaries, reconciliation of medications dispensed and returned, and inspection of returned medication containers at each follow-up visit.

Attendance at scheduled conventional therapy sessions and study visits will be documented throughout the intervention period. Caregivers will also be interviewed regarding adherence to home-based behavioral recommendations. Participants demonstrating suboptimal adherence will receive additional counseling and reinforcement regarding the importance of treatment compliance.

Telephone reminders between scheduled visits will be used to encourage adherence, identify missed doses or therapy sessions, address caregiver concerns, and facilitate timely follow-up. An interdisciplinary team comprising Ayurvedic physicians, pediatricians, psychologists, and study coordinators will review participants’ progress at each visit and implement appropriate measures to optimize protocol adherence and participant retention.

Participants may continue to receive routine medical treatment for conditions unrelated to ASD during the study period. However, the initiation of additional pharmacological interventions specifically intended to modify the core symptoms of ASD will not be permitted after enrollment.

If clinically indicated, investigators may prescribe supportive treatment that is considered necessary for participant safety and routine medical care. All concomitant medications and therapies, including the indication, dosage, frequency, route of administration, and duration of treatment, will be recorded in the case report form (CRF) and reviewed throughout the study to identify potential protocol deviations or confounding factors.

Study treatment may be discontinued in the event of a serious adverse event, clinically significant intolerance, participant withdrawal of consent, or persistent nonadherence to the study protocol. Temporary dose interruption or modification may be considered for minor adverse events (AEs) at the investigator’s discretion, provided participant safety is maintained. All treatment modifications, discontinuations, and their underlying reasons will be documented in the CRFs.

Participation in the study is entirely voluntary. Parents or legal guardians may withdraw their child from the study at any time without affecting future medical care or access to conventional therapy. Participants who withdraw from the study will discontinue the investigational intervention, and the reason for withdrawal, when available, will be documented.

The sample size for this study was calculated based on the primary outcome measure, the ATEC score. Using G*Power software (Heinrich Heine University Düsseldorf), the required sample size was estimated with ATEC as the primary end point. A previous study on children with autism reported mean ATEC scores of 78.70 (SD 21.49) in the control group and 60.40 (SD 23.57) in the treatment group [13]. Based on these values, a sample size of 40 participants per group (total N=80) was required to detect a moderate-to-large effect size (Cohen d ≥0.81), with a 2-tailed α of .05% and 90% power, using an independent t test. This includes an adjustment for an anticipated 20% dropout rate.

Following confirmation of eligibility and completion of baseline assessments, the study coordinator will open the next envelope in sequence to assign the participant to the respective study group.

The study is open-label. Due to the nature of the intervention, participant and investigator blinding is not feasible. However, outcome assessments will be performed using standardized, validated instruments administered according to predefined procedures, and statistical analyses will be conducted using coded datasets to minimize assessment and analytical bias.

Outcome measures will be collected at baseline (T0) and subsequently during the follow-up (T1-T6) visits at 30-day intervals. Periodic evaluation of clinical changes and facilitation of safety monitoring throughout the study will be possible through these scheduled assessments. The primary end point of the study is the change in the ATEC. The secondary end points include changes in the ISAA, WISC-IV, VABS (Daily Living Skills domain), and QoLA for parents. These selected outcome measures were chosen based on published evidence supporting their widespread use in ASD research and clinical practice. Collectively, these instruments provide a comprehensive, multidimensional assessment of cognitive and adaptive functioning in children with ASD, along with a measure of the impact on parents’ quality of life (Table 2). However, a separate psychometric validation of these instruments within this study population was not performed. Additionally, inflammatory biomarkers (hs-CRP, IL-6, and TNF-α) will be evaluated as exploratory mechanistic outcomes to investigate potential modulation of immune and inflammatory pathways implicated in ASD following adjunctive Ayurvedic therapy. Vitamin D, vitamin B12, and serum iron will be measured as part of the baseline clinical and biochemical characterization of the study population and for exploratory analyses. The trial is not designed to evaluate correction of nutritional deficiencies as a therapeutic mechanism.

The study participants may derive potential benefits from the integrative approach in terms of improvements in intellectual and adaptive functioning.

The safety of the study intervention is assessed by documenting any AEs at each scheduled visit. All AEs occurring during the study period are recorded and monitored in accordance with GCP guidelines. Safety evaluations also include hematological and biochemical investigations, as specified.

Participants will attend 7 scheduled visits over 6 months: one baseline visit for screening, enrollment, and initial assessments and 6 follow-up visits (days 30, 60, 90, 120, 150, and 180) for intervention, outcome assessments, and laboratory investigations. A 7-day window period will be allowed for each visit, and parents/guardians will report any AEs immediately (Figure 1).

Baseline demographic, clinical, laboratory, and outcome assessment data will be recorded in standardized CRFs by trained study personnel. Data collected during follow-up visits, including treatment adherence, concomitant medications, AEs, and outcome measures, will be entered promptly into the study database.

All study records will be reviewed for completeness and accuracy before data entry. Double data entry, periodic data verification, and quality-control procedures will be implemented to minimize transcription errors and ensure data integrity. Data queries and discrepancies will be resolved through source document verification.

Participant confidentiality will be maintained by assigning a unique study identification number to each participant. Personal identifiers will be stored separately from study data, and access to identifiable information will be restricted to authorized study personnel. Electronic data will be maintained in password-protected computers with regular data backups, while hard-copy records will be stored in locked cabinets within secured research facilities.

Study documents will be retained in accordance with institutional policies and applicable regulatory requirements upon completion of the trial.

ASD is a complex neurodevelopmental condition that affects multiple domains of functioning, including social communication, behavior, cognition, and adaptive skills. Although multidisciplinary conventional interventions remain the cornerstone of management, many children continue to experience persistent functional limitations despite prolonged therapy. Consequently, there is increasing interest in adjunctive interventions that target multiple biological and behavioral pathways while complementing established therapeutic approaches.

This protocol describes a randomized, open-label, active-controlled clinical trial designed to evaluate the efficacy of an integrative treatment approach combining Saraswata Ghrita and Ayush Manas with conventional therapy for children with ASD. Unlike previous studies that have primarily focused on behavioral outcomes, the present trial adopts a multidimensional evaluation strategy incorporating behavioral symptoms, intellectual functioning, adaptive behavior, caregiver quality of life, and exploratory inflammatory biomarkers. This comprehensive assessment is intended to provide a broader understanding of the potential clinical and biological effects of adjunctive Ayurvedic therapy [32].

The inclusion of inflammatory biomarkers, including hs-CRP, IL-6, and TNF-α, is based on growing evidence suggesting that immune dysregulation and neuroinflammation may contribute to ASD pathophysiology in a subset of individuals. Although these biomarkers are not intended as diagnostic or surrogate end points, their assessment may provide preliminary insights into biological mechanisms associated with treatment response and help inform future mechanistic studies [33-36].

The study also incorporates validated instruments to assess multiple dimensions of ASD, including autism severity, cognitive performance, adaptive behavior, and caregiver quality of life. This multidimensional outcome framework is expected to provide a more comprehensive evaluation of intervention effects than reliance on behavioral measures alone.

This study has several methodological strengths. It is among the few RCTs evaluating Ayurveda as an adjunct to conventional therapy for children with ASD. The protocol follows SPIRIT recommendations and incorporates standardized eligibility criteria, predefined outcome measures, and structured follow-up assessments to enhance methodological rigor.

Another strength is the integration of validated behavioral, cognitive, adaptive, caregiver-reported, laboratory, and safety outcomes. Simultaneous evaluation of these complementary domains enables a comprehensive assessment of treatment effects while maintaining participant safety throughout the study.

Treatment adherence is supported through standardized caregiver education, medication diaries, drug accountability procedures, scheduled follow-up visits, and regular communication with participating families. These measures are expected to improve intervention fidelity and data quality.

Owing to the nature of the interventions, participant and investigator blinding was not feasible. This may have introduced the potential for performance and observer bias, which is acknowledged as a limitation of the study. However, standardized assessment procedures and predefined statistical analyses have been incorporated to minimize bias.

The study is being conducted at a single coordinating center with collaboration from selected autism therapy centers, which may limit the generalizability of the findings to broader populations. In addition, variability in the intensity and frequency of conventional therapies across participants may influence treatment outcomes despite careful documentation of concomitant interventions.

The exploratory biomarker analysis is intended to generate hypotheses regarding potential biological mechanisms rather than establish causal relationships. Consequently, any observed biomarker changes should be interpreted cautiously and confirmed in future adequately powered mechanistic studies.

The present study did not include a separate psychometric validation of the outcome measures, particularly the ATEC, in the study population prior to trial implementation. Although all assessment tools used in the study have established validity and reliability in previous research, their psychometric properties were not specifically evaluated in this cohort. Future studies should consider formal psychometric validation, including assessment of internal consistency, test-retest reliability, and construct validity, to further strengthen the interpretation and generalizability of the findings.

The findings of this trial will contribute to the limited evidence base regarding integrative management of ASD. If the intervention demonstrates acceptable safety and promising clinical benefits, the results will support the design of larger multicenter RCTs with longer follow-up periods and broader participant populations.

Future studies may also incorporate advanced neuroimaging, genomic, metabolomic, and microbiome analyses to further elucidate the biological mechanisms underlying treatment response and identify predictors of therapeutic benefit. Such investigations could facilitate the development of more individualized, integrative treatment strategies for children with ASD.

The study makes a significant step toward the evidence-based integration of Ayurveda with conventional therapy in managing ASD. By integrating validated assessments of behavioral symptoms, intellectual functioning, adaptive behavior, caregiver quality of life, and exploratory inflammatory biomarkers, the study adopts a comprehensive approach to evaluating both the clinical and potential biological effects of the intervention. The findings of the trial are expected to contribute to the limited high-quality evidence regarding the role of Ayurveda as an adjunct to conventional ASD management. If shown to be safe and beneficial, this integrative approach may support the development of evidence-based multidisciplinary treatment strategies and inform future multicenter clinical trials investigating Ayurvedic interventions for neurodevelopmental disorders.