What is the evidence and dose for leucovorin for autism in pediatric patients?

Comment by InpharmD Researcher

Current evidence, including meta-analyses of randomized trials, suggests leucovorin (folinic acid) at 0.5–2 mg/kg/day (max 50 mg/day) may be beneficial in improving core autism symptoms in children with folate-related abnormalities. Significant benefits include reduced stereotyped behaviors, improved communication (medium-to-large effect sizes), and overall symptom improvement (67% response in deficient subgroups). However, the quality of studies were low, primarily due to small sample size with reliance on subjective scoring. Side effects (e.g., agitation, insomnia) are typically mild. Optimal effects may require long-term use (1–2 years).

Background

A 2025 systematic review and meta-analysis evaluated the efficacy of folinic acid in alleviating autism spectrum disorder (ASD) symptoms in children. This meta-analysis synthesized data from two double-blind randomized placebo-controlled trials involving 103 participants. The analysis adhered to the PRISMA guidelines and employed a fixed-effects model due to the lack of observed heterogeneity (I²= 9%) among the included studies. The meta-analysis revealed that the administration of folinic acid at a dose of 2 mg/kg potentially improved symptoms of ASD, as observed through a reduction in the Aberrant Behavior Checklist (ABC) scores. The pooled mean difference was -0.66 with a 95% confidence interval ranging from -1.22 to -0.10, indicating statistical significance (p= 0.02). Particularly, significant improvements were noted in the stereotyped behavior subscale, with a mean difference of -1.60 (95% CI: -3.03 to -0.17, p= 0.03). Despite these promising findings, the small sample size and limited number of studies warrant further research to confirm the therapeutic benefits of folinic acid in children with ASD. [1]

A 2021 systematic review and meta-analysis examined leucovorin treatment for ASD in children. Among 21 included studies, the general dose range was 0.5 to 2.5 mg/kg/day orally, with most studies using doses between 0.5-2 mg/kg/day (maximum 50 mg/day). The strongest evidence came from blinded, placebo-controlled studies which demonstrated that leucovorin significantly improved communication with medium-to-large effect sizes, and showed positive effects on core ASD symptoms and associated behaviors like attention and stereotypy with large effect sizes. For children with ASD and comorbid cerebral folate deficiency, leucovorin was particularly effective with response rates exceeding 75% for treating ataxia, pyramidal signs, and epilepsy, while also improving overall ASD symptoms (67% response rate), irritability (58%), and movement disorders (47%). The treatment was generally well-tolerated with mild adverse effects, most commonly including excitement/agitation (11.7%), aggression (9.5%), insomnia (8.5%), and increased tantrums (6.2%). The authors concluded that leucovorin appears safe and effective for improving core and associated ASD symptoms, with some studies suggesting that younger children and those positive for folate receptor alpha autoantibodies may show more robust improvements, and that treatment effects may require 1-2 years to reach maximum benefit. [2]

References:

[1] Soetedjo FA, Kristijanto JAF, Durry FD. Folinic acid and autism spectrum disorder in children: A systematic review and meta-analysis of two double-blind randomized placebo-controlled trials. Aceh Nutri J. 2025;10(1):194. doi:10.30867/action.v10i1.1743
[2] Rossignol DA, Frye RE. Cerebral Folate Deficiency, Folate Receptor Alpha Autoantibodies and Leucovorin (Folinic Acid) Treatment in Autism Spectrum Disorders: A Systematic Review and Meta-Analysis. J Pers Med. 2021;11(11):1141. Published 2021 Nov 3. doi:10.3390/jpm11111141
Literature Review

A search of the published medical literature revealed 5 studies investigating the researchable question:

What is the evidence and dose for leucovorin for autism in pediatric patients?

Level of evidence

C - Multiple studies with limitations or conflicting results  Read more→


Please see Tables 1-5 for your response.

Efficacy of oral folinic acid supplementation in children with autism spectrum disorder: a randomized double-blind, placebo-controlled trial
Design

Randomized double-blind, placebo-controlled trial

N= 80
Objective To compare changes in Childhood Autism Rating Scale (CARS) scores in children with ASD aged 2–10 years, among folinic acid and placebo groups at 24 weeks, in comparison with baseline
Study Groups

Folinic acid group (n= 40)

Placebo group (n= 40)
Inclusion Criteria Children with ASD (meeting DSM-V criteria) aged 2–10 years; on antipsychotic medications with no changes in the 8 weeks before screening; parents willing to comply with study protocols
Exclusion Criteria Severe gastroesophageal reflux, chronic hepatic or renal problems, medications affecting serum folate levels, known genetic conditions linked to folate metabolism, recent clinical seizure within 6 months, receiving other complementary treatments
Methods Participants in the folinic acid group received oral folinic acid at 2 mg/kg daily (50 mg daily maximum dose) for 24 weeks, while the placebo group received a placebo tablet daily. Both groups received standard care including behavioral interventions like ABA and sensory integration therapy. Assessments were conducted at baseline and after 24 weeks.
Duration January 2022 to January 2024
Outcome Measures

Primary: Change in CARS score at 24 weeks from baseline

Secondary: Changes in Child Behavior Checklist (CBCL) total score, internalizing and externalizing scores, CSHQ score, social quotient (SQ) by VSMS, sensory processing issues by SP-2
Baseline Characteristics   Folinic acid group (n= 40) Control group (n= 40)
Age (years) 5.2 ± 1.8 5.3 ± 1.9
Gender - Male 37 (92%) 38 (95%)
Socioeconomic status - Lower 11 (27%) 12 (30%)
Residence - Rural 5 (12%) 4 (10%)
Use of atypical antipsychotics 16 (40%) 15 (37%)
CARS score 34.5 ± 3.9 35.1 ± 4.3
CBCL total score 69.8 ± 11.4 68.5 ± 10.7
CBCL internalizing score 54.6 ± 10.9 55.2 ± 11.3
CBCL externalizing score 15.3 ± 8.5 13.7 ± 8.4
Number of patients with significant sensory processing abnormalities 29 (72%) 27 (68%)
CSHQ score 38.5 ± 6.9 37.9 ± 7.2
Social quotient 63.6 ± 11.8 64.7 ± 12.6
Serum folic acid level (ng/ml) 11.1 ± 5.2 10.9 ± 5.4
Serum folate receptor autoantibody level (ng/ml) 317.4 ± 149.2 323.8 ± 153.4
High levels of serum folate receptor autoantibody level 32 (80%) 33 (82%)
Results   Folinic acid group (n= 40) Control group (n= 40) p-Value
CARS score 3.6 ± 0.8 2.4 ± 0.7 <0.001
CBCL total score 19.7 ± 9.5 12.6 ± 8.4 <0.001
CBCL internalizing score 15.4 ± 7.8 8.5 ± 5.7 <0.001
CBCL externalizing score 4.3 ± 2.9 4.1 ± 2.7 0.75
Number of patients with significant sensory processing abnormalities 17 (42%) 18 (45%) 0.94
CSHQ score 4.6 ± 3.8 3.7 ± 3.1 0.24
Social quotient 3.4 ± 3.1 3.5 ± 2.9 0.91
Adverse Events No adverse reactions were reported in either group
Study Author Conclusions Oral folinic acid supplementation is effective and safe in improving ASD symptoms, with more pronounced benefits in children with high titers of folate receptor autoantibodies.
Critique

The study demonstrates the efficacy of folinic acid in improving ASD symptoms, particularly in those with high folate receptor autoantibody titers. However, the reliance on parent-reported outcomes may introduce bias, and the study's findings may not be generalizable beyond the specific population studied. Additionally, the placebo effect in ASD research can be significant, potentially affecting the results.

 

References:

Panda PK, Sharawat IK, Saha S, Gupta D, Palayullakandi A, Meena K. Efficacy of oral folinic acid supplementation in children with autism spectrum disorder: a randomized double-blind, placebo-controlled trial. Eur J Pediatr. 2024;183(11):4827-4835. doi:10.1007/s00431-024-05762-6

Improving Outcome in Infantile Autism with Folate Receptor Autoimmunity and Nutritional Derangements: A Self-Controlled Trial
Design

Self-controlled therapeutic trial

N= 82
Objective To assess the impact of correcting nutritional deficiencies and treating folate receptor alpha autoimmunity on the outcomes of children with infantile autism
Study Groups

Treated group (n= 82)

Untreated reference group (n= 84)
Inclusion Criteria Children diagnosed with nonsyndromic infantile autism without genetic abnormalities, brain abnormalities, intractable epilepsy, or recognizable genetic syndromes
Exclusion Criteria Children with underlying syndromes or genetic causes, such as Rubenstein-Taybi syndrome, MECP2 duplication, and specific microdeletions or microduplications
Methods

Participants underwent routine blood tests for CBC, iron, vitamins, coenzyme Q10, metals, and trace elements. Serum FRα autoantibodies were assessed. Nutritional derangements were treated with supplements, and high-dose folinic acid (0.5 to 1 mg/kg/day) was added if FRα autoantibodies were positive. The Childhood Autism Rating Scale (CARS) was used to monitor progress over two years.
Duration Two years
Outcome Measures

Primary: Change in Childhood Autism Rating Scale (CARS) score

Secondary: Correlation of treatment response with FRα autoantibody titers and parental antibody status
Baseline Characteristics   Treated group (n= 82) Untreated group (n= 84)
Age, mean ± SD 4.4 ± 2.3 years 4.45 ± 2.62 years
Male:Female ratio 4.8:1 5.46:1
FRα autoantibodies in children 75.6% 71.4%
FRα autoantibodies in mothers 34.1% 30.8%
FRα autoantibodies in fathers 29.4% 27.4%
Results   Treated group (n= 82) Untreated group (n= 84) p-value
CARS score at baseline, mean ± SD 41.34 ± 6.47 41.92 ± 5.4 --
CARS score after 2 years, mean ± SD 34.35 ± 6.25 41.92 ± 5.4 <0.0001
Complete recovery (CARS < 30) 17/82 (20.7%) 0/84 (0%) --
Adverse Events

No specific adverse events reported related to the treatment protocol
Study Author Conclusions

Correction of nutritional deficiencies combined with high-dose folinic acid improved outcomes for autism. The presence of maternal FRα antibodies or antibodies in both parents may warrant folinic acid intervention before conception and during pregnancy.
Critique

The study's self-controlled design allowed for individualized treatment, but the small sample size and lack of randomization may limit the generalizability of the findings. The study did not differentiate the effects of correcting nutritional deficiencies from those of folinic acid treatment, making it difficult to attribute improvements to a specific intervention.

 

References:

Ramaekers VT, Sequeira JM, DiDuca M, et al. Improving Outcome in Infantile Autism with Folate Receptor Autoimmunity and Nutritional Derangements: A Self-Controlled Trial. Autism Res Treat. 2019;2019:7486431. Published 2019 Jun 18. doi:10.1155/2019/7486431

Folinic acid improves verbal communication in children with autism and language impairment: a randomized double-blind placebo-controlled trial
Design

Randomized double-blind placebo-controlled trial

N=48
Objective To determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment
Study Groups

Folinic acid (n=23)

Placebo (n=25)
Inclusion Criteria Age 3–14 years; documentation of language impairment; unchanged complementary, traditional, behavioral and education therapy 8 weeks prior to enrollment; intention to maintain ongoing therapies constant throughout the trial
Exclusion Criteria Antipsychotic medications; supplementation exceeding the recommended daily allowance; prematurity; uncontrolled gastroesophageal reflux; history of liver or kidney disease; drugs known to affect folate metabolism; profound sensory deficits; well-defined genetic syndromes; genetic mutations known to significantly affect folate-associated pathways; brain malformations or damage found on MRI; ongoing therapies that could interfere with the study; a clinical seizure within the last 6 months; moderate-to-severe irritability or self-abusive behavior on the aberrant behavior checklist
Methods

Participants were randomized to receive 12 weeks of high-dose folinic acid (2 mg/kg per day, maximum 50 mg per day) or placebo. Language, developmental, and behavioral assessments were conducted at baseline and after 12 weeks. Parent and teacher questionnaires were collected at baseline, 6 weeks, and 12 weeks. Adverse events were monitored every 3 weeks.
Duration 12 weeks
Outcome Measures

Primary: Improvement in verbal communication

Secondary: Improvement in folate receptor-α autoantibody (FRAA)
Baseline Characteristics   Folinic acid (n= 23) Placebo (n= 25)
Age, mean (s.d.), years months 7y 7 m (3y 6 m) 7y 2 m (2y 10 m)
Males, N (%) 18 (78) 20 (80)
Folate receptor autoantibody positive, N (%) 13 (57) 18 (72)
Blocking titer (pmol ml −1), mean (s.d.) 0.08 (0.20) 0.06 (0.15)
Binding titer (pmol ml −1), mean (s.d.) 0.39 (0.74) 0.61 (0.73)
Glutathione redox ratio, mean (s.d.) 9.21 (2.40) 9.09 (1.72)
Folate (ng ml −1), mean (s.d.) 17.15 (3.41) 17.79 (2.42)
B12 (pg ml −1), mean (s.d.) 859.79 (447.54) 725.39 (368.06)
s.d.= standard deviation
Results   Folinic acid (n=23) Placebo (n=25) p-value
Improvement in verbal communication (standardized points) 5.7 (1.0,10.4) - 0.02
Effect size (Cohen’s d) 0.70 - -
FRAA positive improvement (standardized points) 7.3 (1.4,13.2) - 0.02
Effect size (Cohen’s d) for FRAA positive 0.91 - -
Adverse Events

There was no significant difference in adverse effects between treatment groups. Common adverse events included excitement or agitation, insomnia, increased motor activity, and restlessness
Study Author Conclusions

In this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.
Critique

The study demonstrated significant improvement in verbal communication with folinic acid treatment, especially in FRAA-positive children. However, the small sample size and single-site design limit the generalizability of the findings. Further studies with larger sample sizes and longer durations are needed to confirm these results and assess long-term safety.

 

References:

Frye RE, Slattery J, Delhey L, et al. Folinic acid improves verbal communication in children with autism and language impairment: a randomized double-blind placebo-controlled trial. Mol Psychiatry. 2018;23(2):247-256. doi:10.1038/mp.2016.168

Folinic acid as a treatment for autism in children: A within-subjects open-label study on safety and efficacy
Design

Open-label pre-post treatment within-subjects design study

N= 10
Objective To determine the safety, feasibility, and efficacy of oral folinic acid in improving communication and behavior in autistic children
Study Groups All participants (n= 10)
Inclusion Criteria Age 3 years 0 months to 10 years 11 months; weight 10–30 kg; confirmed diagnosis of autism based on DSM criteria; clinical evidence of language impairment; stable on regular intervention and medications for at least 6 months before recruitment
Exclusion Criteria Known allergy or intolerance to folinic acid; pre-existing supplementation with folinic acid or undefined folates; vitamin B12 deficiency; concurrent use of certain medications; genetic/metabolic abnormalities; history of epilepsy; sensory or motor impairment; active malignancy; participation in another clinical trial for autism within the preceding 6 months
Methods Participants underwent a control period (0–12 weeks) with standard care, followed by a treatment period (12–24 weeks) with oral folinic acid at 2 mg/kg/day. Safety evaluations, standardized assessments, and eye-gaze tracking were conducted at T = 0, 12, and 24 weeks.
Duration November 2022 to July 2023
Outcome Measures

Pervasive Developmental Disorder Behavior Inventory (PDDBI) score (188-item caregiver completed rating scale on a 4-point Likert scale from 0; total T-score of 50 reflects the average symptom score that an autistic child would show, while total T-scores below 40 indicate low to minimal autism symptoms)
Baseline Characteristics   All patients (n= 10)
Age in years, median (range) 6.5 (4 to 8)
Gender - Male 9
Gender - Female 1
Ethnicity - Chinese 7
Ethnicity - Indian 3
Weight in kg at recruitment, mean (SD) 21.0 (3.2)
IQ standard scores - Non-verbal IQ 69.0 (26.8)
IQ standard scores - Verbal IQ 56.2 (17.5)
IQ standard scores - Full scale IQ 60.5 (22.5)
Diagnosis of intellectual disability 7
FRAA status - Negative 4
FRAA status - Positive binding antibody 5
FRAA status - Positive blocking antibody 1
Actual daily dose of folinic acid administered in mg/kg/day, mean (SD) 1.95 (0.13)
Results   Score at T = 0w, mean ± SD Score at T = 12w, mean ± SD Score at T = 24w, mean ± SD Change score during control period, mean ± SD Change score during treatment period, mean ± SD p-value
PDDBI Total score (TOTAL) 49.3 ± 6.65 49.2 ± 8.89 44.6 ± 6.19 -0.10 ± 6.10 -4.60 ± 6.38 0.103
PDDBI Expressive language (EXPRESS) 35.0 ± 20.8 34.3 ± 22.1 39.8 ± 20.8 -0.70 ± 6.29 5.50 ± 5.50 0.010
PDDBI Learning, memory, receptive language (LMRL) 21.1 ± 9.29 19.1 ± 9.33 26.3 ± 7.38 -2.00 ± 4.47 7.20 ± 6.05 0.002
Adverse Events No adverse events reported. Mild adverse events such as hyperactivity/impulsivity, problems with RRBs, aggression/irritability, confusion, mood swings, sleeping problems, rashes, anxiety/low mood, fevers with no cause, serious or recurrent infections, loss of appetite, self-harm, and diarrhea were noted but not deemed related to folinic acid consumption
Study Author Conclusions Folinic acid is a safe and feasible potential treatment for autism, and results from this pilot justify the need for a larger placebo-controlled trial.
Critique The study is limited by its small sample size and open-label design, which may introduce bias. The lack of a placebo control limits the ability to attribute improvements solely to folinic acid. However, the study successfully demonstrated safety and feasibility, providing a basis for larger trials.

 

References:

Wong CM, Tan CS, Koh HC, et al. Folinic acid as a treatment for autism in children: A within-subjects open-label study on safety and efficacy. Int J Dev Neurosci. 2025;85(1):e10402. doi:10.1002/jdn.10402

Safety and Efficacy of High-Dose Folinic Acid in Children with Autism: The Impact of Folate Metabolism Gene Polymorphisms
Design

12-week randomized clinical trial

N= 80
Objective To evaluate the safety and efficacy of high-dose folinic acid intervention in Chinese children with ASD and explore the association between folate metabolism gene polymorphisms and efficacy
Study Groups

Intervention group (n= 50)

Control group (n= 30)
Inclusion Criteria Children aged 3 to 6 years diagnosed with ASD by two specialized clinicians according to DSM-5 criteria
Exclusion Criteria Presence of severe gastrointestinal symptoms, congenital heart disease, liver or kidney disease, epilepsy, brain malformations, taking antipsychotic medications, previous or current use of folate-related drugs, mitochondrial diseases, other genetic metabolic disorders, severe mental or neurological conditions
Methods Participants in the intervention group received folinic acid at a dose of 2 mg/kg per day (maximum 50 mg/day), given orally in two divided doses for 12 weeks. Efficacy was measured using the PEP-3 at baseline and 12 weeks. Genotyping for MTHFR C677T, MTHFR A1298C, MTR A2756G, and MTRR A66G was performed in the intervention group.
Duration August 2021 to August 2023
Outcome Measures

Improvements in Psycho-Educational Profile, Third Edition (PEP-3) scores which measure three main composites for communication ability
Baseline Characteristics   Control Group (n= 27) Intervention Group (n= 49)
Age at enrollment, months 51.30 (12.94) 51.27 (9.75)
Gender - Female 5 (18.5%) 7 (14.3%)
Birth weight, kg 3.42 (0.43) 3.21 (0.42)
Results   Control Group Change Intervention Group Change p-value
Social reciprocity 0.33 (1.71) 1.53 (1.98) 0.010

Among the different characteristics analyzed using the PEP-3 scoring system, only social reciprocity demonstrated a statistically significant benefit compared to control. Furthermore, children with MTHFR A1298C or MTRR A66G mutations demonstrated greater improvements in various developmental domains compared to wild type.
Adverse Events No significant adverse effects were observed during the intervention
Study Author Conclusions High-dose folinic acid may be a promising intervention for children with ASD, and its efficacy is associated with folate metabolism gene polymorphisms. High-dose folinic acid intervention may promote better neurodevelopmental outcomes by alleviating folate metabolism abnormalities caused by single or combined mutations in folate metabolism genes.
Critique

The open-label design may introduce bias, and the small sample size for some genotypes may limit the strength of causal inference. Furthermore, only social reciprocity demonstrated a statistical benefit while other parameters like language, motor function, mood, and behaviors were mixed. Therefore, in spite of the author's conclusion, there is no clearly observable benefit on the macro scale and follow-up studies are required.

 

References:

Zhang C, Chen Y, Hou F, et al. Safety and Efficacy of High-Dose Folinic Acid in Children with Autism: The Impact of Folate Metabolism Gene Polymorphisms. Nutrients. 2025;17(9):1602. Published 2025 May 7. doi:10.3390/nu17091602