Potential mechanisms and research progress of animal-assisted interventions for autism spectrum disorder

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with onset in early childhood. Conventional behavioral interventions are limited by poor adherence and variable efficacy. Animal-assisted intervention (AAI) creates a low-stress therapeutic environment through positive human-animal interaction and has become an important adjunctive approach for ASD. This review summarizes the clinical evidence, underlying mechanisms, and current limitations of AAI. Clinical evidence indicates that dog-assisted and equine-assisted interventions can improve core symptoms and common comorbidities. However, treatment effects are highly heterogeneous, effect sizes vary widely, several high-quality studies have reported no significant benefit, and the overall level of evidence remains low. Mechanistically, AAI may act through multiple pathways involving neurobiological regulation, physiological and immune modulation, psychological and behavioral processes, and sensorimotor integration. To date, only hypothalamic-pituitary-adrenal axis regulation, autonomic nervous system modulation, and social brain network remodeling have gained preliminary direct support. Synaptic plasticity, immune-inflammatory regulation, and mitochondrial metabolism remain largely hypothetical. Optimal mechanisms may differ across ASD subgroups, requiring stratified intervention based on age, cognitive level, and sensory characteristics. In addition, stress in therapy animals is not only an ethical concern but also a confounding factor that may affect attribution of treatment effects. Current evidence is limited by the lack of high-quality randomized controlled trials, heterogeneous intervention assessment, and insufficient mechanistic evidence. Future research should prioritize large-scale randomized controlled trials, distinguish direct from indirect evidence, include animal stress in statistical analyses, and develop personalized intervention strategies to advance standardized, evidence-based AAI practice.

Key Words: Animal-assisted intervention; Autism spectrum disorder; Neurobiological mechanisms; Clinical rehabilitation; Social communication

Core Tip: This review evaluates clinical evidence and mechanisms of animal-assisted intervention (AAI) for autism spectrum disorder. Clinically, dog- or equine-assisted interventions improve symptoms and comorbidities, but efficacy is highly heterogeneous with variable effect sizes, and several high-quality studies demonstrate no significant benefit, indicating low-quality evidence. Mechanistically, AAI regulates hypothalamic-pituitary-adrenal axis, oxytocin, social brain networks, autonomic function, and sensorimotor integration. However, direct evidence is limited to neuroendocrine responses, heart rate variability, and electroencephalographic changes; synaptic plasticity, immune regulation, and mitochondrial metabolism remain theoretical. Optimal mechanisms differ by subgroup, highlighting stratification by age, cognition, and sensory characteristics. Methodologically, animal stress is a confounder affecting interaction quality. Future trials are supposed to include animal stress as a covariate and establish stratified frameworks for standardized, evidence-based AAI practice.

INTRODUCTION

Autism spectrum disorder (ASD) is a neurodevelopmental condition with onset in infancy and early childhood. It is characterized by deficits in social communication, restricted and repetitive behaviors, and narrow interests[1,2]. Motor delay, emotional dysregulation, and impaired adaptive behavior are common, with substantial impact on lifelong development and family quality of life[3,4]. Global prevalence is about 1%, with a steadily increasing burden in children, making ASD a major public health concern[5,6]. Current clinical management relies mainly on non-pharmacological behavioral interventions. Conventional training is limited by poor adherence, marked heterogeneity in outcomes, and difficulty in simultaneously improving multiple core symptom domains[7,8]. Safe, well-tolerated adjunctive strategies that address broader symptom profiles are therefore needed.

Animal-assisted intervention (AAI) is a targeted rehabilitation approach delivered by trained professionals using trained animals as therapeutic mediators[1,9]. It includes therapeutic horseback riding (THR) and equine-assisted activities and therapies (EAAT), dog-assisted therapy (DAT), and dolphin-assisted intervention[1,2,5,10]. By promoting positive human-animal interaction, AAI creates a low-stress and highly accepting therapeutic environment with good engagement and safety. It has been widely used in neurodevelopmental rehabilitation and serves as an important complement to conventional interventions, offering new directions for ASD management[11-14]. Multiple systematic reviews and meta-analyses have shown that AAI significantly improves social, communication, and motor functions in children with ASD[5,15,16]. THR improves social interaction and communication, as well as fine and gross motor skills, balance, and coordination[3,15]. EAAT shows broad benefits in social, communication, motor skills, and quality of life[5]. DAT enhances social communication and emotional regulation in children with ASD[1]. In addition, AAI reduces physiological stress indicators such as cortisol and improves heart rate variability (HRV), contributing to symptom improvement[17-19].

However, most studies focus on efficacy, while molecular and neural mechanisms remain insufficiently defined. Limitations also include small sample sizes, heterogeneous intervention protocols, and variable evidence quality[2,16,20], which restrict clinical standardization. This review summarizes advances in AAI for ASD, analyzes potential mechanisms, and outlines limitations and future directions to support clinical translation and mechanism-driven research.

LITERATURE SEARCH STRATEGY

To ensure a comprehensive and non-biased integration of available literature on the clinical progress and potential mechanisms of AAIs for ASD, a rigorous literature search was systematically conducted across major biomedical databases, including PubMed, Web of Science, CNKY, PsycINFO, and Scopus, from database inception up to May 2026. The search strategy utilized a combination of Medical Subject Headings terms and relevant free-text keywords, structured into three primary thematic blocks using Boolean operators: (1) Target population: “Autism spectrum disorder” OR “autism” OR “ASD”; (2) Intervention: “Animal-assisted intervention” OR “animal-assisted therapy” OR “equine-assisted therapy” OR “canine-assisted intervention” OR “therapeutic horseback riding” OR “hippotherapy” OR “pet therapy”; and (3) Mechanisms/outcomes: “Mechanism” OR “neurobiology” OR “oxytocin” OR “cortisol” OR “synaptic plasticity” OR “immune” OR “inflammation” OR “brain connectivity” OR “clinical efficacy”.

The inclusion criteria were restricted to: (1) Peer-reviewed original research articles, randomized controlled trials (RCTs), quasi-experimental studies, and high-quality systematic reviews; (2) Studies evaluating the therapeutic outcomes or investigating biological/psychological mechanisms of AAI in individuals with ASD or relevant animal models; and (3) Articles published in English. Document types such as conference abstracts, dissertations, case reports with sample sizes less than three, and non-peer-reviewed preprints were excluded to maintain methodological rigor. Two independent reviewers screened titles, abstracts, and subsequently full-text articles to minimize potential selection bias, with any discrepancies resolved through consensus or consultation with a third senior reviewer (Figure 1).

Figure 1 PRISMA 2020 flow diagram of the literature identification, screening and inclusion process. A systematic search of five databases (PubMed, Web of Science, Scopus, PsycINFO, and CNKI) identified 2381 records. After removing 503 duplicates, 1878 records were screened by title and abstract, of which 1245 were excluded. Of the 633 reports sought for retrieval, 9 were not accessible. The remaining 624 full-text reports were assessed for eligibility, and 340 were excluded primarily for inconsistent intervention definitions or irrelevant outcomes. The final review included 284 studies, comprising 67 randomized controlled trials, 96 non-randomized or pre-post studies, 42 cross-sectional or observational studies, 23 qualitative studies, and 56 systematic reviews or meta-analyses. Numbers represent the number of records at each stage. ASD: Autism spectrum disorder.

MAJOR TYPES AND ADVANCES OF AAI FOR ASD

DAT

DAT is one of the most extensively studied forms of AAI and has been associated with improvements in social communication, emotional regulation, and attachment-related outcomes in individuals with ASD[1,9,21]. Several studies have shown that DAT increases eye contact, verbal and non-verbal communication, and social initiation in children with ASD[22,23]. Improvements have also been reported in communication motivation, behavioral regulation, and participation in community activities[24]. A controlled crossover study found that DAT improved adaptive social communication skills in young children with ASD, with benefits persisting beyond the intervention period[25]. Compared with robotic dogs, live therapy dogs appear to elicit greater social responsiveness in children with severe language impairments and may promote social initiation and reciprocal interaction more effectively[19,26].

DAT may also contribute to emotional regulation and stress reduction. Previous studies have reported lower perceived stress levels in adults with ASD, accompanied by improvements in social awareness and communication[27]. In children, interactions with therapy dogs have been associated with increased emotional security, greater engagement in therapeutic activities, and reductions in self-injurious and repetitive behaviors[28,29]. Even a single session with a therapy dog has been reported to reduce heart-rate reactivity and attenuate task-related stress responses[30]. Enhanced behavioral synchrony between children and therapy dogs may further contribute to emotional regulation[31]. In addition, anxiety reduction associated with DAT may indirectly improve sleep quality in children with ASD[32,33]. The supportive and non-judgmental nature of therapy dogs may also facilitate attachment formation. Interactions with therapy dogs can help children recognize emotional cues, encourage positive affect and imitative behaviors, and promote participation in therapeutic activities[19,28,30,34,35].

Hippotherapy and EAAT

Equine-assisted interventions, including THR and hippotherapy, represent a major category of AAI. These interventions have attracted particular attention because of their potential benefits for sensorimotor integration and executive functioning in individuals with ASD[5,36].

The rhythmic, three-dimensional movement of a horse generates continuous vestibular and proprioceptive input that is transmitted through the rider’s pelvis and trunk[3,37,38]. This unique form of sensory stimulation may contribute to improvements in motor control and sensory processing. A meta-analysis reported moderate-to-large effects of THR on social and communication outcomes in children with ASD[15]. Other reviews have similarly found that EAAT is associated with improvements in social functioning, communication, and motor skills[3,5,39].

Regarding motor performance, hippotherapy has been linked to gains in balance, coordination, muscle strength, and overall motor function[3,40-43]. These benefits may extend to daily activities and enhance functional participation. During riding sessions, children continuously adjust their posture to maintain balance, which may support the development of postural control and sensory integration[39,44].

Equine-assisted interventions may also influence executive functioning. The dynamic and interactive nature of riding activities requires planning, attention, and behavioral flexibility[45,46]. Studies have reported improvements in executive function measures following standardized hippotherapy programs, accompanied by changes in frontal neural activity and functional connectivity[45]. THR has also been associated with better planning and task-execution abilities in children with ASD[46].

Dolphin and other AAIs

Research on dolphin-assisted and other less common forms of AAI remains limited. Nevertheless, these interventions may provide additional insight into the diverse mechanisms through which human-animal interactions influence ASD-related outcomes[2,47].

Dolphin-assisted therapy has been reported to improve communication-related behaviors in children with ASD. A RCT found that children receiving dolphin-assisted therapy showed greater increases in vocalizations and gesture use than those receiving conventional interventions[10]. Neurophysiological studies have also suggested that dolphin-assisted therapy may influence functional brain connectivity, as reflected by changes in alpha-band coherence and delta-band activity[48]. In addition, improvements in therapist-child interaction synchrony have been linked to gains in language development[49]. However, the available evidence remains limited by small sample sizes and the potential influence of environmental novelty[10,48].

Studies involving smaller animals, such as cats and guinea pigs, are comparatively rare[2,50]. Children with ASD have been reported to show increased visual attention during interactions with cats, possibly because feline social signals are less intrusive and easier to tolerate[51]. Although affectionate behaviors displayed by cats may vary according to symptom severity, the emotional value attributed to cats by children with ASD appears comparable to that reported by typically developing children[50]. Guinea pigs have been observed to display more positive social behaviors during initial interactions with children with ASD, followed by reductions in feeding and resting behaviors, suggesting sensitivity to social and environmental cues[52]. Collectively, these studies indicate that interactions between small animals and children with ASD represent a dynamic and reciprocal process. They also highlight the importance of considering animal welfare as a central component of both AAI research and clinical practice[50,52].

CLINICAL PROGRESS OF AAIS IN IMPROVING ASD

Effects of AAI on core ASD symptoms

Improvements in social communication and social interaction: AAI demonstrates the most consistent benefits in alleviating social communication impairments (Table 1). Meta-analyses have shown that THR and EAAT significantly improve social and communication skills in children with ASD[5,15]. Galvany-López et al[1] reported that dog-assisted interventions enhance key behaviors such as eye contact and social initiation. Although a systematic review by Sissons et al[2], covering 38 studies, rated the overall evidence as low to moderate, improvements across multiple functional domains were still observed.

Table 1 Summary of clinical outcomes of animal-assisted intervention in autism spectrum disorder.

Clinical outcome	Dog-assisted intervention	Therapeutic horseback riding and equine-assisted interventions	Dolphin-assisted intervention	Other animal-assisted interventions
Social communication and social interaction	[9,19,22,24-30,34,35,66,76,77,79-82,143]	[4,15,41,43,46,53-59,64,65,73,78,84-103,105,106]	[10,48,49]	[50-52,109,112]
Repetitive and rigid behaviors	[22,24,29]	[53,56-58,65,90,95,96,100,102]	-
Sensory processing and sensory integration	-	[39,41,60,94,106]	-	-
Emotional regulation	[9,24,28,30,35,66,79,81,111]	[45,46,62-65,78,85,97,100,103-107]	-	[50,109,110]
Motor function	[68,69]	[3,4,40-43,46,65,67,86,94,108]	-	[61]
Sleep disturbances and hyperactivity	[1,24,27,72]	[53,63,73,103-105]	-	-
Caregiver stress and negative emotional burden	[18,27,77,83]	[65,74]	-	-
Family functioning and quality of care	[76,77]	[62,64,65,78,91]	-	-

High-quality RCTs provide more robust evidence. Gabriels et al[53] found that a 10-week THR program significantly improved Social Responsiveness Scale-Second Edition total scores and Vineland Socialization scores in children with ASD, with effects sustained at 3-month follow-up. Bass et al[54] further confirmed improvements in Social Skills Rating System total and subscale scores following THR. In addition, dog training interventions[25] and equine-assisted occupational therapy[55] have been shown to enhance adaptive social communication and social motivation. However, effect sizes for social outcomes are consistently higher in RCTs than in non-randomized studies, suggesting that findings from non-RCT designs should be interpreted cautiously[15].

Reduction in repetitive behaviors and behavioral rigidity: AAI may effectively reduce repetitive behaviors, irritability, hyperactivity, and other maladaptive behaviors in ASD. An RCT by Gabriels et al[53] demonstrated that THR significantly reduced scores on the Aberrant Behavior Checklist (ABC) irritability subscale, with effects maintained at follow-up. Lanning et al[56] reported significant reductions in ABC hyperactivity scores following equine-assisted activities. Single-subject studies also indicate reductions in stereotyped behaviors, with partial generalization to classroom settings[57]. Longitudinal parent-reported data[58] suggest that equine interventions reduce ASD symptom severity and hyperactivity. Dog-assisted early interventions have shown moderate to large effect sizes in reducing repetitive behaviors[22]. Qualitative findings from parents consistently indicate that THR improves emotional regulation and reduces behavioral problems[59].

Improvement in sensory processing and integration: Sensory processing abnormalities are common in ASD, and AAI shows potential in improving sensory integration. A 12-week equine therapy program improved coping-related scores on the Vineland Adaptive Behavior Scales, suggesting enhanced sensory integration[41,60]. At the motor-sensory level, a 16-week equine-assisted intervention improved balance and coordination[40]. Elephant-assisted therapy[61] and hippotherapy[39] have also been associated with improved postural control and balance. These findings suggest that rhythmic vestibular and tactile inputs provided through AAI may facilitate multisensory integration, thereby improving adaptive behavior and functional outcomes[3,5,39].

Effects of AAI on comorbid conditions in ASD

Improvement in emotional regulation: AAI has demonstrated significant benefits in reducing emotional dysregulation, including anxiety, irritability, and impulsivity. A 10-week THR program significantly reduced irritability scores on the ABC in children with ASD[53] and promoted generalization of emotional regulation skills[62]. Even a single session of DAT reduced salivary cortisol levels in adults with ASD, while longer-term interventions improved social awareness and communication[18]. Equine-assisted activities have similarly been shown to reduce problem behaviors and improve impulse control[63]. Qualitative studies further suggest that THR enhances emotional regulation, self-confidence, and self-esteem[64,65]. Dog-assisted interventions promote prosocial behavior and emotional regulation in both verbal and non-verbal children with ASD, with stronger emotional engagement observed in non-verbal participants[66].

Improvement in motor function: Motor impairments are among the most common comorbidities in ASD, and equine-assisted interventions provide the strongest evidence for motor benefits[3,5,20,39]. A 12-week THR program significantly improved total motor function, fine and gross motor skills, balance, coordination, and muscle strength[67]. A 16-week equine-assisted intervention increased Bruininks-Oseretsky Test of Motor Proficiency, Second Edition total scores by 10-15 standard points[40]. Hippotherapy has also been associated with improvements in executive function and resting-state electroencephalogram (EEG) markers, with notable effect sizes[45]. Even a single 30-minute session can enhance postural control[39], while elephant-assisted therapy may also improve balance[61]. Dog-assisted interventions have been shown to improve gait and posture in adults with ASD and promote physical activity and health outcomes in children[68,69].

Alleviation of sleep disturbances and hyperactivity: Sleep disturbances affect approximately 16.9% of children with ASD[70]. A network meta-analysis indicated that physical activity interventions such as equine therapy may yield greater improvements in sleep than melatonin or behavioral interventions[32]. Equine-assisted therapy has also been shown to reduce hyperactivity and improve behavioral regulation[63,71]. Dog-assisted interventions can reduce restlessness, pre-sleep agitation, and sleep-onset difficulties[1,24,27,72]. THR significantly reduces irritability and hyperactivity[53], with no reported adverse events and good adherence[67,73]. Overall, AAI appears effective in alleviating both sleep disturbances and hyperactive behaviors in children with ASD.

Indirect benefits of AAI for caregivers of individuals with ASD

Reduction in caregiver stress and negative emotions: Caregivers of children with ASD commonly experience high levels of stress, anxiety, and depression[74]. A 16-week THR program significantly reduced caregiver stress scores on the Depression Anxiety Stress Scales-21-item version, alleviating emotional burden related to disease management and resource constraints[74]. EAAT not only promote psychosocial development in children but also enhance caregiver well-being and parent-child bonding[64]. THR reduces caregiver negative emotions by improving child symptoms and adaptive behaviors[65]. Systematic reviews further confirm that improvements in physical, social, and emotional functioning in children positively influence family functioning regardless of AAI modality[75]. Five sessions of DAT (animal-assisted therapy) can indirectly reduce caregiver burden by improving communication, behavioral regulation, and community participation in children with ASD[24]. Overall, AAI reduces caregiver psychological distress primarily through improvements in core ASD symptoms.

Improvement in family functioning and care quality: AAI contributes to improved family functioning and care quality at the systemic level. EAAT strengthens family cohesion and parent-parent interaction, enhancing familial intimacy[64]. DAT improves self-awareness and enjoyment in individuals with ASD, thereby enhancing family interaction quality[76]. Therapy dogs provide emotional security and facilitate interactions with therapists, whose professional competence is critical for intervention efficacy[28]. Assistance dog placement has been associated with reduced ASD severity and improved family well-being, although sample sizes remain limited[77]. Improvements in self-concept, self-regulation, and social functioning induced by equine interventions may extend through ecological and “ripple” effects to caregivers and the broader family system[78]. In summary, AAI enhances family care quality and overall well-being by reducing caregiver burden and strengthening parent-child interaction and family cohesion (Table 1)[79-112].

Inconsistent findings and heterogeneous effects of AAI in ASD: Although AAI has shown an overall positive trend in improving the core symptoms of ASD, the current evidence is marked by inconsistent findings and substantial heterogeneity in treatment effects, limiting its translation into routine clinical practice. Several systematic reviews and meta-analyses have reported improvements in social functioning, communication, and adaptive behavior. However, effect sizes vary markedly across studies, and some trials have found no significant benefit[16,113,114]. For example, Sissons et al[2] reviewed 38 studies and found that although most reported improvement in at least one domain of social functioning, effect sizes differed considerably, some studies showed no significant improvement, and the overall quality of evidence was rated as low to moderate. Similar inconsistency is evident in meta-analyses. Dimolareva and Dunn[16] analyzed 16 studies and found only small effects of AAI on social interaction and communication in children with ASD, with no clear dose-response relationship. Trzmiel et al[115] likewise reported no statistically significant overall effect.

These heterogeneous findings largely reflect methodological limitations and the lack of standardized intervention protocols across the field. Many studies are limited by small sample sizes, the absence of randomized controlled designs, non-blinded outcome assessment, and short follow-up periods[2,20,116,117], all of which increase the risk of bias. In a systematic review of 20 studies, Davis et al[113] concluded that methodological weaknesses and conflicting findings preclude firm conclusions regarding the effectiveness of AAI for children with ASD. Intervention protocols are also highly variable, differing in animal species (e.g., horses, dogs, and dolphins), intervention frequency, session duration, and total treatment period[12,118,119]. The absence of consistent terminology and standardized implementation frameworks further limits comparisons across studies[9,119]. O'Haire[116] and Nieforth et al[118] both noted that although the number of publications has doubled over the past five years, methodological rigor has improved only modestly. Accordingly, there is broad consensus that large-scale RCTs with standardized intervention protocols, blinded outcome assessment, and long-term follow-up are essential before AAI can be established as an evidence-based intervention for ASD[2,15,16,20,113,118,120].

POTENTIAL MECHANISMS UNDERLYING THE EFFECTS OF AAI IN ASD

Neurobiological mechanisms

Regulation of the hypothalamic-pituitary-adrenal axis - reduction of cortisol levels: Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a key neuroendocrine basis of abnormal stress responses in ASD. AAI may reduce physiological stress and anxiety by suppressing excessive HPA-axis activation and lowering cortisol levels[74]. A single session of animal-assisted therapy significantly reduces cortisol concentrations in adults with ASD[18]. Equine-assisted interventions markedly decrease salivary cortisol levels in children with ASD, accompanied by improvements in social attitudes[99,105]. THR influences the association between cortisol levels and irritability, suggesting a close relationship between HPA-axis reactivity and behavioral symptoms[105,121]. Hippotherapy also improves emotional status in highly stressed individuals with ASD and tends to reduce salivary α-amylase levels[107]. These findings suggest that non-threatening rhythmic sensory stimuli, including horseback movement and gentle dog contact, may contribute to HPA-axis homeostasis[19,22,74] (Figure 2).

Figure 2 Proposed mechanistic framework of animal-assisted intervention for autism spectrum disorder. Animal-assisted intervention may exert its therapeutic effects through four interconnected pathways: Neurobiological regulation (hypothalamic-pituitary-adrenal axis modulation, oxytocin system activation, brain network remodeling, and neuroplasticity enhancement), physiological and immunometabolic modulation (autonomic nervous system regulation, cytokine balance restoration, and mitochondrial function improvement), psychological-behavioral facilitation (emotional security, social motivation, behavioral synchrony, and self-control), and sensorimotor integration (vestibular, proprioceptive, and multisensory input processing). These pathways converge to improve core autism spectrum disorder symptoms. HPA: Hypothalamic-pituitary-adrenal; mPFC: Medial prefrontal cortex; BDNF: Brain-derived neurotrophic factor; mTOR: Mammalian target of rapamycin; AAI: Animal-assisted intervention; HRV: Heart rate variability; IL: Interleukin; TNF-α: Tumor necrosis factor-α; INN: Interneuron network; ATP: Adenosine triphosphate; ASD: Autism spectrum disorder.

Activation of the oxytocin system - enhancement of social motivation and prosocial behavior: The oxytocin (OXT) system regulates social motivation, attachment, and prosocial behavior. OXT dysfunction is considered an important contributor to social deficits in ASD[122-124]. AAI may activate OXT-related pathways[125]. Equine-assisted interventions can alter progesterone levels, an OXT precursor and regulatory hormone, indirectly supporting OXT involvement in treatment effects[18,99]. Improvements in social behavior following AAI are consistent with the social reward function of OXT[14]. Studies have reported increased emotional bonding, eye contact, and willingness to engage in social interaction among children with ASD[17,29]. These benefits may generalize to interactions with family pets[62]. Current evidence suggests that activation of the OXT system may be an important mechanism underlying the effects of AAI on social motivation and prosocial behavior[126,127].

Functional brain reorganization and neurophysiological modulation - optimization of prefrontal and social brain networks: Neuroimaging studies suggest that animal interaction enhances prefrontal cortical activation and may influence brain networks involved in social cognition[128]. Combined with evidence of improved social functioning, these findings indicate that AAI may promote reorganization of prefrontal-social brain network connectivity[19,129,130]. Functional magnetic resonance imaging studies have shown that AAI-based social cognitive training increases medial prefrontal cortex activity in children with ASD, and this increase correlates with social improvement[131]. As a core component of the social brain network, the medial prefrontal cortex is involved in theory of mind and self-referential processing. Quantitative EEG studies demonstrate that dolphin-assisted therapy enhances interhemispheric coherence in the prefrontal alpha band and stabilizes posterior frontal delta activity, indicating improved functional connectivity[48]. EEG studies in adolescents with ASD have also shown increased occipito-temporal coherence following social interventions, accompanied by more frequent social engagement[132]. AAI may therefore alleviate core social deficits by promoting integration and synchronization within social brain networks[131,133-135].

Neuroplasticity, synaptic remodeling, and activation of the mirror neuron system: As an environmental enrichment intervention, AAI provides integrated sensory, motor, and emotional stimulation that may enhance neuroplasticity, improve synaptic function, and activate the mirror neuron system (MNS)[15,118,135]. Animal interaction can induce glia-mediated synaptogenesis, dendritic remodeling, and strengthened neuronal connectivity[135], providing a neural basis for improvements in motor and executive functions[136-139]. Synaptic excitation-inhibition imbalance and abnormal neurotrophic signaling are core pathological features of ASD[140,141]. Exercise-based AAI, particularly horseback riding, may regulate brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) pathways, thereby promoting synaptic remodeling and neuronal survival[142]. Animal studies have similarly shown that environmental enrichment increases BDNF expression and enhances synaptic plasticity[135].

MNS dysfunction is closely linked to deficits in social imitation and empathy in ASD. Live dogs significantly increase spontaneous imitation in children with severe ASD, outperforming robotic dogs, likely because their natural and predictable behavioral cues are more readily encoded and matched by the MNS[143]. They also improve treatment compliance while reducing emotional stress and heart rate reactivity[30]. Deficits in motor coordination are strongly associated with the severity of social symptoms in children with ASD, suggesting a shared neural basis for motor and social functions[144]. Collectively, these findings indicate that AAI promotes action understanding, intention attribution, and social learning by engaging rhythmic action observation and execution, activating the MNS and parietal-premotor circuits, and facilitating concurrent neuroplastic changes[145]. Behavioral evidence directly supports MNS activation[143], whereas molecular regulation through the BDNF/mTOR pathway remains hypothetical[135,142].

Physiological and immunometabolic mechanisms

Regulation of the autonomic nervous system - improvement in HRV: Autonomic nervous system dysfunction is common in ASD and is closely associated with abnormal stress responses and social impairment[146-148]. A single session of animal-assisted therapy can rapidly reduce cortisol levels and provide acute stress relief[18]. Hippotherapy significantly increases HRV sample entropy in children with neurological disorders, indicating improved autonomic regulation[44]. A 30-minute horseback riding session immediately alters center-of-pressure displacement in children with ASD and improves postural control and autonomic responses under challenging conditions[39]. Only live dogs have been shown to increase HRV and attenuate reductions in parasympathetic activity during social tasks[19]. Hippotherapy also improves executive function and stabilizes resting-state frontal delta, theta, alpha, and beta oscillations, suggesting coordinated regulation between autonomic and central nervous systems[45]. Subjective experiences such as relaxation and enjoyment reported during animal-assisted therapy are consistent with improvements in autonomic function[76]. AAI may therefore reduce physiological stress through parasympathetic activation, cortisol reduction, and HRV enhancement, ultimately improving emotional and behavioral regulation[147,148].

Modulation of chronic neuroinflammation and cytokine dysregulation: Accumulating evidence indicates that chronic neuroinflammation and immune dysregulation are important pathological mechanisms in ASD[149-151]. Children with ASD commonly exhibit abnormal immune activation characterized by Th1/Th17 polarization and elevated plasma levels of interleukin-2 (IL-2), IL-12, and IL-17[152]. Increased frequency of the IL-6-174G/C GG genotype and elevated serum IL-6 levels further support the involvement of IL-6-mediated inflammatory signaling in ASD pathogenesis[153]. Extracellular vesicles derived from individuals with ASD can induce peripheral lymphocytes to release pro-inflammatory cytokines such as interferon-γ, thereby maintaining chronic low-grade inflammation[154]. Elevated serum BDNF levels have also been reported in children with ASD and are associated with symptom severity[155], whereas tumor necrosis factor-α (TNF-α) levels are negatively correlated with social functioning[156]. Persistent microglial activation and increased levels of IL-1β, IL-6, and TNF-α are recognized features of ASD-related neuroinflammation and are closely linked to social communication deficits and repetitive behaviors[149-151,157].

Direct evidence that AAI regulates inflammatory cytokines remains limited. However, studies of exercise and environmental enrichment show that these interventions promote neuroplasticity through modulation of BDNF/mTOR signaling, suppression of pro-inflammatory cytokines, and restoration of immune homeostasis[142,158]. As an environmental enrichment intervention combining physical activity, social interaction, and emotional bonding, AAI may alleviate neuroinflammation and immune imbalance by regulating the Th1/Th17 axis and reducing pro-inflammatory cytokines such as IL-6 and TNF-α, thereby improving social and emotional functioning in ASD[118,126,135]. At present, direct evidence for cytokine regulation by AAI is still scarce, and these mechanisms are largely extrapolated from studies of exercise interventions and environmental enrichment[142,158].

Restoration of mitochondrial function and metabolic homeostasis: Mitochondrial dysfunction is considered one of the most common metabolic abnormalities in ASD, characterized by impaired energy metabolism, increased oxidative stress, and abnormal lactate metabolism[159,160]. A study involving 88 children with ASD found significantly elevated serum fibroblast growth factor-21 levels, accompanied by altered lactate metabolism and reduced levels of essential and branched-chain amino acids, indicating a mitochondrial stress state[161]. A recent meta-analysis further confirmed abnormalities in lactate, pyruvate, adenosine triphosphate, and mitochondrial DNA-related biomarkers in individuals with ASD, which were closely associated with deficits in social interaction, cognition, and motor function[162]. ASD is also accompanied by dysregulation of the mitochondrial unfolded protein response and impaired oxidative phosphorylation, affecting neuronal energy supply and synaptic plasticity[160,163]. Multi-omics studies have shown that ASD risk genes are significantly enriched in extracellular matrix homeostasis, lipid, and sphingolipid metabolic pathways, suggesting a synergistic interaction between metabolic dysfunction and genetic susceptibility[164].

Although direct evidence that AAI improves mitochondrial function is lacking, exercise interventions have been shown to promote mitochondrial biogenesis, enhance oxidative phosphorylation efficiency, and regulate BDNF signaling pathways[142,158]. As an environmental enrichment intervention integrating physical activity and social interaction, equine-assisted and dog-assisted activities within AAI may indirectly promote neural recovery in ASD by improving energy metabolic homeostasis, enhancing mitochondrial function, and supporting neuroplasticity[67,165-167]. Currently, direct evidence for mitochondrial improvement through AAI remains unavailable, and these mechanisms are inferred indirectly from exercise-based interventions[142,158,165].

Psychological and behavioral mechanisms

Establishing a sense of security and reducing anxiety: Individuals with ASD commonly experience social anxiety and heightened psychological defensiveness. Animals provide a non-threatening social context that rapidly reduces stress responses[1,18,168]. AAI alleviates anxiety through calming and attentional-shifting effects[14]. Therapy dogs provide emotional security and promote relaxation and acceptance during treatment[28,169], while equine-assisted interventions reduce physiological stress markers and improve emotional states in children with ASD[99]. A single DAT session can reduce cortisol levels in adults with ASD and induce positive emotional experiences, including relaxation and enjoyment[18,76]. The sense of security established during intervention may generalize to home settings, leading to reductions in anxiety and behavioral problems[24,78]. Through non-judgmental companionship and soothing tactile contact, AAI creates a low-threat social environment and may represent an important mechanism for reducing social anxiety in ASD[118,168].

Enhancing social motivation and social initiation: Reduced social motivation is a core feature of ASD. AAI may function as a social catalyst that promotes spontaneous social engagement[170]. Studies show that AAI significantly increases social interaction frequency in children with ASD, with small-to-moderate effect sizes[16,116]. DAT increases social initiation and outperforms non-living stimuli such as toys and robotic dogs[19,143]. Equine-assisted interventions increase interactions with peers, adults, and horses while enhancing attention toward therapists and group activities[22,100,109]. These benefits can extend across settings. THR has been associated with greater affection toward family pets and improved social functioning at school[62,88]. By capitalizing on the intrinsic appeal of animals, AAI may activate social motivation and sustain active social participation in individuals with ASD[168].

Behavioral synchrony and positive behavior shaping: AAI strengthens behavioral synchrony between individuals with ASD and both animals and therapists, creating conditions favorable for social skill acquisition[126,171,172]. DAT improves movement synchrony between children and therapy dogs[31], whereas dolphin-assisted therapy enhances verbal interaction synchrony between children and therapists[49]. Improved synchrony may facilitate positive behavioral change. THR reduces irritability, hyperactivity, and repetitive behaviors while improving adaptive functioning[4,43,58,90,105]. Dog-training interventions improve social communication scores, decrease aggression and self-stimulatory behaviors, and enhance compliance and task completion[25,30,113]. The predictable responses and immediate feedback provided by animals may help individuals with ASD interpret social cues and develop more appropriate social behaviors[28,173].

Strengthening emotional regulation and self-control: Emotional dysregulation is a common comorbidity in ASD. AAI offers unique advantages for emotion recognition, expression, and regulation[149,174-176]. DAT improves emotional functioning, emotional regulation, and self-esteem in children with ASD[1,176]. It also reduces anxiety-related autonomic arousal and cortisol secretion, supporting emotional stability[18,99]. Equine-assisted interventions increase positive affect and reduce negative emotional expression[100]. Even a single equine-assisted session can improve emotional states in adolescents and young adults with ASD, with greater benefits observed in highly stressed individuals[107]. Parent reports indicate reductions in emotional outbursts and improvements in self-soothing abilities following AAI[24,78]. A 16-week THR program significantly enhanced self-control[88]. By acting through both physiological and psychological pathways, AAI strengthens emotional regulation and self-control, supporting broader social adaptation in ASD[64,176-178].

Sensorimotor integration mechanisms: Sensory processing abnormalities and motor impairments are highly prevalent in ASD and often interact to restrict social participation[179,180]. AAI, particularly equine-assisted interventions, may improve sensorimotor integration through multidimensional proprioceptive, vestibular, and tactile stimulation[67,181].

Proprioceptive and vestibular input during equine-assisted interventions: The therapeutic effects of THR and hippotherapy largely derive from the horse’s three-dimensional rhythmic gait, which provides continuous vestibular and proprioceptive input to the rider[3,37,67,181]. THR significantly improves gross motor skills, balance, mobility, and object-control abilities in children with ASD, with large effect sizes reported[166]. After 12 weeks of THR, Bruininks-Oseretsky Test of Motor Proficiency, Second Edition scores for overall motor performance, fine and gross motor skills, balance, coordination, and muscle strength are significantly higher than those of controls[67]. Hippotherapy can immediately alter center-of-pressure displacement, indicating adaptive modulation of proprioceptive processing[39]. Multi-elephant therapeutic programs have also improved balance control in individuals with ASD[61]. Beyond motor outcomes, hippotherapy enhances adaptive behavior, participation, and sensory regulation[41,60]. It also stabilizes resting-state frontal δ, θ, α, and β oscillations, changes associated with improved executive function and suggestive of central nervous system adaptation to vestibular-proprioceptive stimulation[45]. Although some reviews consider the evidence for perceptual-motor outcomes to be limited[71], multiple high-quality studies support beneficial effects[115]. The unique kinematic properties of horse movement provide a sensory training environment that is difficult to replicate by other interventions[3,43,182].

Multisensory input from other animals and sensory integration enhancement: Dogs, dolphins, and other animals may improve sensory integration through distinct multimodal sensory inputs[47,49,66,126,183]. The deep-pressure tactile stimulation provided by therapy dogs exerts calming effects and reduces physiological stress responses[14,18]. Compared with toys or robotic dogs, live dogs are more effective in eliciting spontaneous imitation, improving emotional regulation, and enhancing compliance in children with severe ASD[30,143]. Only live dogs have been shown to increase HRV and buffer parasympathetic withdrawal during social tasks[19]. Dolphin-assisted therapy combines aquatic tactile stimulation, high-frequency acoustic input, and proprioceptive feedback generated during swimming. These inputs improve vocalization frequency and gesture use in children with ASD[10]. Dolphin interaction also increases interhemispheric coherence in the prefrontal α band, suggesting that sensory stimulation may contribute to functional brain network plasticity[48]. Collectively, different animals provide distinct sensory modalities that support multidimensional sensory integration training, thereby improving environmental information processing and adaptive behavior in individuals with ASD[153,184].

Based on the four mechanistic pathways described above, Table 2 summarizes the core evidence supporting each pathway and classifies evidence levels according to direct evidence and indirect/theoretical inference. Current findings indicate that AAI has preliminary direct support at the levels of neuroendocrine regulation, autonomic function, brain activity, and psychological behavior. However, deeper molecular mechanisms, including synaptic plasticity, immune-inflammatory regulation, and mitochondrial metabolism, remain largely based on indirect evidence and require further validation.

Table 2 Potential mechanisms of animal-assisted intervention in improving autism spectrum disorder: Evidence sources and classification.

Mechanistic dimension	Specific pathway	Evidence type	Key findings and corresponding effects	Ref.
Neurobiological mechanisms	HPA axis regulation (cortisol reduction)	Direct evidence	AAI (e.g., equine-assisted and dog-assisted interventions) significantly reduces salivary cortisol levels in individuals with ASD and improves stress and irritability symptoms	[18,99,105,121]
	Oxytocin system activation	Theoretical inference	Based on changes in oxytocin-related hormonal responses and social reward functions, AAI is proposed to enhance social motivation and prosocial behaviors through oxytocin system activation	[14,18,99,125-127]
	Prefrontal cortex and social brain network remodeling	Direct evidence	Neuroimaging and EEG studies indicate that dog-assisted therapy and social training increase prefrontal activation and improve α/δ-band connectivity	[48,128,131,132]
	Synaptic remodeling and mirror neuron system activation	Theoretical inference	Dynamic behaviors of live animals may activate the mirror neuron system; exercise-based AAI may enhance the BDNF/mTOR pathway, promoting synaptic remodeling and action understanding	[15,30,135,142,143,145]
Physiological, immune, and metabolic mechanisms	Autonomic nervous system regulation (HRV enhancement)	Direct evidence	Horse riding and interaction with live dogs significantly increase HRV sample entropy, enhance parasympathetic activity, and stabilize emotional states	[19,39,44,45,76]
	Chronic neuroinflammation and cytokine regulation	Theoretical inference	Based on Th1/Th17 polarization and abnormal pro-inflammatory cytokines (IL-6, TNF-α) in ASD, AAI is proposed to suppress inflammatory responses through environmental enrichment effects	[135,142,149,152,156,158,161]
	Mitochondrial function and metabolic homeostasis restoration	Theoretical inference	Based on mitochondrial stress and abnormal lactate metabolism in ASD, combined with exercise-related effects, AAI may indirectly promote mitochondrial biogenesis and energy metabolic balance	[67,142,159-162,165]
Psychological and behavioral mechanisms	Establishment of safety and anxiety reduction	Direct evidence	Therapy dogs and horses provide non-judgmental companionship and deep-pressure tactile stimulation, rapidly reducing emotional stress, fear, and anxiety	[1,18,24,28,76,78,99,168]
	Enhancement of social motivation and active social engagement	Direct evidence	Animals act as “social catalysts”, significantly increasing spontaneous eye contact, verbal/non-verbal communication, and social initiation in children with ASD	[19,22,100,109,143,116,170]
	Behavioral synchrony and positive behavior shaping	Direct evidence	AAI improves motor and verbal synchrony between individuals with ASD and animals/therapists, reducing irritability, hyperactivity, and repetitive behaviors	[25,30,31,49,58,105,113]
	Consolidation of emotional regulation and self-control	Direct evidence	AAI enhances self-control and self-soothing abilities, reduces emotional outbursts, and a 16-week equine intervention significantly improves self-control scores	[1,18,24,64,88,99,107,176]
Sensorimotor integration mechanisms	Vestibular and proprioceptive input (equine-assisted intervention)	Direct evidence	Three-dimensional rhythmic movement from horses provides continuous vestibular and proprioceptive stimulation, significantly improving BOT-2 scores, balance, gross and fine motor skills, and postural control	[3,15,37,39,40,61,67,166,181,182]
	Multimodal sensory input and sensory integration	Direct evidence	Deep-pressure tactile stimulation from dogs and tactile/acoustic stimulation from dolphins improve multisensory integration and enhance adaptive responses to environmental stimuli	[10,14,18,19,30,48,135]

HPA: Hypothalamic-pituitary-adrenal; AAI: Animal-assisted intervention; ASD: Autism spectrum disorder; EEG: Electroencephalogram; mTOR: Mammalian target of rapamycin; BDNF: Brain-derived neurotrophic factor; HRV: Heart rate variability; IL: Interleukin; TNF-α: Tumor necrosis factor-α; BOT-2: Bruininks-Oseretsky Test-2^nd Edition.

Modulatory effects of population and intervention heterogeneity on therapeutic mechanisms: The high heterogeneity of ASD determines substantial subgroup differences in the mechanisms underlying AAI effects. Understanding how different variables regulate the contribution of specific mechanistic pathways is a theoretical basis for designing personalized AAI strategies.

Participant characteristics play a critical role in mechanism selection. Regarding ASD severity, children with severe ASD often show greater deficits in social motivation[1,170]. Live dogs significantly enhance spontaneous imitation through activation of the MNS, with stronger effects than robotic dogs[143], suggesting that these children may preferentially benefit from canine-mediated social brain network remodeling[19,131]. In contrast, individuals with high-functioning ASD may rely more on psychological and behavioral mechanisms, including social motivation enhancement and behavioral synchrony[16,116]. For sensory characteristics, children with tactile defensiveness may respond better to the HPA axis regulatory effects of deep-pressure tactile stimulation from dogs, showing greater reductions in cortisol levels[18,28]. Children with vestibular-proprioceptive sensitivity may benefit more from the rhythmic oscillatory stimulation provided by equine-assisted intervention through sensorimotor integration pathways[39,67,166]. Regarding cognitive ability, individuals with lower baseline language skills may improve spontaneous imitation through MNS activation[143], whereas those with better cognitive function may rely more on optimization of social brain network connectivity to enhance social abilities[131,138]. In terms of comorbid anxiety, highly stressed subgroups show greater emotional improvement after a single session of equine therapy[107], indicating a specific value of HPA axis regulation in this population. Children with lower anxiety levels may benefit more from autonomic regulation, particularly HRV improvement[19,44]. Age also influences treatment mechanisms. Children exhibit greater brain plasticity, with stronger effects on synaptic remodeling and neural network optimization[135,136,138], whereas adolescents and adults may rely more on emotional regulation and behavioral shaping mechanisms[107].

Intervention characteristics also determine the dominant mechanistic pathways. Dogs enhance safety perception and emotional regulation through deep-pressure tactile stimulation[14,18,28], whereas horses promote sensorimotor integration through dynamic proprioceptive input[3,37,67]. Dolphin-assisted intervention combines aquatic tactile stimulation and high-frequency acoustic signals, facilitating remodeling of prefrontal interhemispheric functional connectivity[48]. Regarding intervention dose, moderate intensity (total duration ≤ 1440 minutes) appears optimal for improving motor function and sensory integration[166,185], suggesting that this dose range may maximize the cumulative effects of sensorimotor pathways. In contrast, psychological and behavioral effects, such as establishing a sense of safety and enhancing social motivation, may emerge rapidly after a single exposure[18,76]. The treatment environment and therapist factors also influence outcomes. Natural outdoor settings may reduce defensive responses in children[64,168] and enhance HPA axis regulation[28,64]. The therapist’s professional guidance directly affects the establishment of behavioral synchrony and positive behavior shaping[28,160]. Parental involvement further facilitates generalization of psychological and behavioral improvements to home settings[24,62,78], while reductions in caregiver negative emotions may indirectly improve family interaction quality[64,65,74].

Overall, optimal response mechanisms differ substantially across ASD subgroups. Future studies should develop stratified models based on these moderating variables[186,187] and match personalized AAI protocols to achieve precision intervention.

METHODOLOGICAL LIMITATIONS OF AAI RESEARCH IN ASD

Although AAI has shown benefits for core symptoms of ASD, the overall methodological quality of the evidence remains limited, restricting both scientific advancement and clinical translation.

Limitations in study design

The lack of high-quality study designs is the most prominent challenge in the AAI field. Large-scale, multicenter RCTs remain scarce, and most studies use non-randomized controlled or quasi-experimental designs, with limited randomization and multicenter validation[2,5,15,20]. Among the 38 studies included by Sissons et al[2], only 11 were RCTs. Similarly, only 8 of 48 studies reviewed by McDaniel Peters and Wood[20] were RCTs. Sample sizes were generally small, with only the THR study by Gabriels et al[53] enrolling 127 participants. Multicenter intervention RCTs are particularly rare[70], substantially reducing the strength and reliability of efficacy evidence[5,90]. Another major limitation is the lack of blinding, placebo controls, and long-term follow-up. Because interventions involve animals, complete blinding of participants, therapists, and parents is difficult to achieve. Blinding of outcome assessors has also not been consistently implemented, increasing the risk of detection bias[2,9,15]. Most studies use waitlist controls or usual care as comparators and lack sham intervention controls, making it difficult to exclude non-specific effects[2,5,9,20]. Only a few studies include 3-6 months follow-up assessments, whereas most evaluate short-term outcomes only, limiting conclusions regarding treatment durability[5,53,95].

Small sample sizes and short intervention periods are also common. Most studies include 20-60 participants, and some pilot studies involve only 8-10 individuals, resulting in insufficient statistical power and increased risks of false-negative or false-positive findings[2,5,9,15,20,34]. Interventions typically last 8-16 weeks, with 1-2 sessions per week and a total duration of 10-20 hours. Although moderate-duration interventions appear to produce better outcomes, the long-term benefits remain unclear[5,15,118,166]. Research involving adolescents, adults, and individuals with low-functioning ASD remains limited, restricting the generalizability of findings[2,166,179].

Moderator variables and heterogeneity

This section focuses on methodological sources of heterogeneity and their impact on evidence integration, rather than subgroup differences in mechanisms. Current findings on AAI for ASD show limited consistency. The major sources of heterogeneity can be systematically examined through three categories of moderators: Participant characteristics, intervention parameters, and social environmental factors. These factors remain major barriers to evidence synthesis and clinical translation.

Participant-related moderators represent the fundamental basis of heterogeneity. Age is a key predictor of effect size differences. Existing studies mainly focus on children with ASD aged 2-12 years, with greater improvements in social function than those observed in adolescents and adults. Age-related differences in neuroplasticity and social development may contribute to these variations in treatment response[2,5,166,179]. Lack of stratification by ASD severity and baseline functioning further increases heterogeneity. Many studies include mainly high-functioning individuals, while those with low functioning and intellectual disability are often excluded. However, children with severe ASD show better adherence and behavioral responses to live dog interventions, suggesting a non-linear relationship between functional level and treatment response[17,30,53,166]. Comorbid conditions also influence intervention effects. Children with comorbid attention-deficit hyperactivity-disorder or anxiety disorders show substantial differences in improvements in hyperactivity and irritability. Higher comorbidity burden is associated with greater baseline symptom fluctuations and lower stability of treatment evaluation[53,56,63,71,188]. In addition, baseline sensory sensitivity directly affects responses to multisensory interventions such as equine and dolphin-assisted therapies, with distinct benefit pathways among sensory response subtypes[39,60,179].

Intervention-related moderators are direct sources of cross-study incomparability. Animal species determine the primary functional outcomes. Dog-assisted interventions primarily improve social communication and emotional regulation[1,9,21], whereas equine-assisted interventions show stronger effects on sensorimotor integration and executive function[3,5,67]. Differences in therapeutic targets limit direct pooling across intervention types. Intervention dose and duration also lack standardized criteria. Most current protocols involve 8-16 weeks of intervention, 1-2 sessions per week. Long-term cumulative effects remain unclear, and uncertainty regarding dose-response relationships further increases heterogeneity[5,15,166,185]. In addition, variations in environmental settings, therapist expertise, and animal training quality may indirectly affect treatment outcomes by altering the quality of human-animal interaction[28,47,100].

Social environmental moderators contribute to differences in treatment generalization. Parental involvement and family support positively predict maintenance of treatment effects. In families with higher participation, reduced caregiver stress may create positive feedback that reinforces improvements in child functioning[64,74,78]. Peer interaction levels in group-based interventions may further enhance social gains and improve skill transfer in group AAI programs such as equine-assisted therapy[46,100].

Insufficient mechanistic evidence

Mechanistic studies of AAI in ASD remain at an early stage, with limited causal validation and in-depth analysis. Basic experimental evidence is lacking. Current studies are mainly based on clinical correlations, reporting changes in indicators such as cortisol, HRV, and EEG connectivity, which cannot establish causal mechanisms[18,44,45,48,99]. The neurobiological basis of behavioral benefits remains unclear. Specific animal models for AAI are lacking, and the core components of human-animal interaction have not been systematically isolated or validated[12,62,135,189,190], leaving placebo effects difficult to exclude. Molecular and cellular mechanisms also remain poorly defined. Studies measuring hormones and inflammatory factors generally involve small samples and heterogeneous results, with no established link between AAI and pathological pathways[18,99,153,155,156]. There is currently no direct evidence for cellular-level regulation involving BDNF dynamics, synaptic plasticity, microglial activation, or mitochondrial function, although these pathways are all implicated in ASD pathophysiology[45,54,160,189,191]. Therefore, the mechanisms underlying AAI remain largely a “black box”.

Animal welfare and gaps in clinical translation

Animal welfare in AAI has been largely overlooked, and its clinical implications have been substantially underestimated. Existing studies often treat animal welfare as an ethical compliance issue only. Few studies have assessed physiological and behavioral stress in therapy horses and dogs, while standardized protocols for health monitoring, rest periods, and withdrawal criteria are generally lacking. The long-term effects of repeated interventions on animal psychological well-being remain unclear[12,13,118,192,193]. From the perspective of reciprocal dynamics in human-animal interactions, therapy animals are not passive intervention tools. Stress, fatigue, and negative emotional states experienced by animals during interventions represent important confounding factors that may directly affect treatment safety and efficacy in children with ASD.

The therapeutic effects of AAI depend on dynamic bidirectional interactions between humans and animals. Animal stress states can directly alter interaction quality. Guinea pigs exposed to sustained interactions with children with ASD show stress-related reductions in feeding and resting behaviors, indicating that animals can perceive child behaviors and generate negative physiological responses[52]. In families of children with severe ASD, cats exhibit significantly fewer affiliative behaviors[50]. Such weakened interaction patterns may directly reduce behavioral synchrony, which is a key mediator of AAI-induced improvements in social communication and emotional regulation[31,49]. In addition, animal stress signals and avoidance behaviors may reduce perceived safety and impair attachment formation in individuals with ASD, weakening the emotional calming effects of AAI and potentially increasing anxiety and repetitive behaviors[28,168].

Current studies rarely include animal workload, stress levels, and behavioral states as control variables. Random fluctuations in animal condition may introduce systematic errors and compromise accurate attribution of treatment effects[13,192]. During long-term interventions, reduced interaction quality caused by chronic animal fatigue may also contribute to the attenuation of effects observed during follow-up assessments[53,95]. Meanwhile, clinical translation and standardization guidelines for AAI remain insufficient. There are no unified evidence-based standards for animal selection, therapist qualifications, or intervention procedures, limiting reproducibility and broader application of research findings[2,9,12,119,194,195]. These limitations substantially hinder the transition of AAI from a complementary intervention to a standardized evidence-based therapy.

FUTURE DIRECTIONS AND PERSPECTIVES

Current evidence indicates that AAI can improve core symptoms of ASD[1,2,5,15]. However, this field remains limited by low methodological quality, unclear mechanisms, heterogeneous intervention protocols, and insufficient standardization. Future research should focus on the following directions.

Large-scale multicenter RCTs with long-term follow-up

Insufficient methodological quality remains a major limitation of AAI research, characterized by small sample sizes, non-randomized designs, lack of active controls, and short observation periods[9,20,114]. Although RCTs have demonstrated that 10-week THR improves social communication, with effects maintained for 3-6 months[53,95], most studies still involve limited samples and poor generalizability[17], and long-term effects remain unclear[4]. Future studies should conduct multicenter, blinded RCTs with more than 100 participants per group, include active controls such as agricultural activities[89], extend follow-up periods beyond 12 months, and determine the long-term efficacy and optimal dose of AAI[32] to improve evidence quality and clinical reliability.

Elucidating molecular and neural mechanisms

The mechanisms underlying AAI remain unclear and require systematic investigation across neuroendocrine, brain network, immune, and metabolic levels. Physiological studies indicate that AAI reduces cortisol levels, regulates progesterone responses, and increases HRV, thereby improving autonomic function[17,18,44,99]. Neuroimaging and EEG studies suggest that AAI optimizes prefrontal neural oscillations and functional connectivity[45,48], enhances medial prefrontal cortex activity, and improves social functioning[128]. Future studies should integrate functional magnetic resonance imaging, magnetoencephalography, EEG, and molecular approaches to clarify how AAI regulates key neural circuits, including the MNS, default mode network, and reward pathways, as well as immune-inflammatory processes[158,189], thereby revealing the “black box” of AAI mechanisms.

Developing personalized AAI programs based on ASD subtypes

The high heterogeneity of ASD limits the effectiveness of a single AAI protocol, highlighting the need for personalized intervention systems. Studies indicate that children respond better to equine-assisted interventions than adolescents and adults[5], while comorbidities, baseline language ability, and functional level also influence treatment outcomes[25,70]. Children with severe ASD show better adherence to live dog interventions[30]. Future studies should develop stratified models based on genetic profiles, neural phenotypes, sensory characteristics, and comorbidity patterns[186,187], clarify the matching relationship between ASD subgroups and specific animals or intervention formats, and achieve precision intervention.

Establishing international standards for implementation, evaluation, and ethics

AAI protocols and outcome measures remain highly heterogeneous, resulting in poor comparability across studies and limiting clinical translation[20,119,196,197]. Existing risk assessment tools for live animal-assisted therapy and animal welfare guidelines have not been widely adopted[12,195]. The concept of synchronized human and animal health assessment under the “one health” framework has yet to be implemented[193]. Future efforts should be led by international organizations to establish core outcome sets, minimum reporting standards, and ethical and welfare guidelines for AAI. Standardized intervention procedures, personnel qualifications, and efficacy assessment systems are needed to improve research reproducibility and the reliability of clinical decision-making[9].

Integrating AAI with intelligent technologies

Live-animal-based AAI is limited by high costs, restricted accessibility, and challenges related to animal welfare. Intelligent technologies may provide innovative alternatives and supplements. Studies have shown that live dogs, social robots, and virtual dolphins can improve social and cognitive functions in individuals with ASD[19,198,199]. Motion-based video games may also produce greater improvements in executive function than conventional exercise[200]. Future approaches should develop hybrid intervention models combining live animals, virtual reality, and social robots. Integration with wearable sensors and artificial intelligence-based emotion recognition technologies may enable real-time feedback and personalized adjustment, overcoming spatial and temporal limitations while improving the accessibility, engagement, and precision of AAI[201-203].

CONCLUSION

This review systematically summarizes the application of AAI in ASD and confirms that dog-assisted and equine-assisted interventions have positive effects on improving core symptoms and comorbidities. However, current evidence also reveals substantial heterogeneity and inconsistent outcomes. Effect sizes vary widely across studies, some high-quality trials fail to demonstrate significant benefits, and the overall certainty of evidence remains low. Therefore, positive findings should be interpreted with caution. At the mechanistic level, AAI may exert effects through multiple pathways, including HPA axis regulation, OXT activation, social brain network remodeling, autonomic regulation, psychological and behavioral processes, and sensorimotor integration. However, among these mechanisms, only HPA axis regulation, HRV improvement, and EEG changes have received preliminary direct support from ASD-AAI studies, whereas deeper mechanisms involving synaptic plasticity, immune-inflammatory regulation, and mitochondrial metabolism remain largely based on indirect theoretical inference. More importantly, optimal response mechanisms differ substantially among ASD subgroups. Children and individuals with severe ASD may benefit more from MNS activation and sensorimotor integration. Individuals with high anxiety or stress levels may preferentially respond to HPA axis regulation, whereas children with tactile defensiveness may rely more on autonomic regulation induced by deep-pressure stimulation from dogs. These findings highlight the need for stratified and personalized AAI strategies based on age, cognitive level, and sensory characteristics.

In addition, animal stress is not only an ethical concern but also an important confounding factor in treatment evaluation. Fluctuations in animal state during bidirectional interactions may directly affect interaction quality and the attribution of outcomes. Future studies should conduct large-scale RCTs to confirm efficacy, clearly distinguish direct evidence from theoretical inference, incorporate animal stress indicators as covariates, and establish stratified intervention frameworks to advance AAI toward standardized, reproducible, evidence-based practice.