Efficacy of folic acid supplementation in the prevention of cardiovascular disease - a systematic review and meta-analysis of randomized controlled trials

Abstract

Background

Cardiovascular disease (CVD) remains the leading global cause of death, with a growing interest in modifiable risk factors such as homocysteine. Elevated homocysteine contributes to atherosclerosis through endothelial dysfunction and oxidative stress. Folic acid, a key cofactor in homocysteine metabolism, has been proposed as a preventive strategy. However, recent studies have provided conflicting evidence regarding its effectiveness in preventing cardiovascular outcomes beyond stroke.

Methods

Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, we conducted a search in multiple databases, including PubMed, Cochrane, Scopus, Web of Science, CINAHL, and EMBASE, and pre-registered on PROSPERO: CRD42024525945. Data were pooled using a random-effects model in R (version 3.4.3), with relative risks (RRs), mean differences (MD) and 95% confidence intervals (CIs) calculated.

Results

Our search yielded a preliminary list of 25,881 articles, with 45 studies that includes 96,962 participants meeting the inclusion criteria. These studies evaluated the effectiveness of folic acid supplementation in preventing cardiovascular disease and related events. The meta-analysis showed that folic acid supplementation reduced the risk of stroke (RR = 0.85, 95% CI: 0.76–0.96, p < 0.01) and modest reduction on overall CVD (RR = 0.95, 95% CI: 0.90–0.99, p = 0.04). No significant effects were found for mortality (RR = 0.98, 95% CI: 0.93–1.02), CHD (RR = 0.98, 95% CI: 0.91–1.06), PAD (RR = 0.94, 95% CI: 0.75–1.17), HDL (MD = 0.31, 95% CI: -0.04–0.65), or LDL (MD = -1.59, 95% CI: -8.82–5.64).

Conclusions

These findings reveal the capability of folic acid supplementation in reducing the risk of stroke and cardiovascular disease. However, no benefit was found for other cardiovascular endpoints. These findings support folic acid’s targeted use in primary prevention strategies, but further research is needed to refine patient selection and clarify its role in broader cardiovascular risk management.

Key points

1. Stroke Risk Reduction: Folic acid supplementation significantly reduces stroke risk but has no significant effect on cardiovascular disease, coronary heart disease, or peripheral arterial disease.

2. No Effect on Lipid Profiles: Folic acid supplementation does not significantly impact high-density lipoprotein (HDL) or low-density lipoprotein (LDL) levels.

3. Study Variability and Bias: There is substantial heterogeneity and some high-risk biases across the studies included, which suggests the need for careful interpretation of the results.

4. Further Research Needed: The findings underscore the necessity for additional research to better understand how folic acid supplementation might more broadly influence cardiovascular health.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40795-025-01178-z.

Introduction

As the leading cause of death worldwide, cardiovascular disease (CVD) is a condition that truncates life expectancy and quality of life for those affected. The number of deaths has increased from 12,3 million to 19,4 million from 1990 to 2021, respectively [1]. A key component of cardiovascular disease is atherosclerosis formation or arterial wall lining. Atherosclerotic formation occurs due to endothelial function impairment and damage to the endothelial lining itself [2]. Homocysteine (Hcy) is generated from S-adenosylhomocysteine (AdoHcy) and metabolized to cystathionine by cystathionine β-synthase (CBS) and to Hcy-thiolactone by methionyl-tRNA synthetase. Hcy-thiolactone, a chemically reactive thioester, modifies protein lysine residues, generating N-homocysteinylated (N-Hcy)-protein. N-Hcy-proteins lose their normal native function and become cytotoxic, autoimmunogenic, proinflammatory, prothrombotic, and proatherogenic. Accumulating evidence, shows that these Hcy metabolites can promote endothelial dysfunction, CVD, and stroke in humans by inducing pro-atherogenic changes in gene expression, upregulating mTOR signaling, and inhibiting autophagy through epigenetic mechanisms involving specific microRNAs, histone demethylase PHF8, and methylated histone H4K20me1. Also, studies show that cystathionine and Hcy-thiolactone are associated with myocardial infarction and ischemic stroke by influencing blood clotting [3]. Thus lowering the levels of homocysteine has been shown to reduce CVD complications such as recurrent stroke risk and peripheral artery disease [4, 5]. However, new studies in medicine drive a new pathway on how homocysteine plasma levels may be reduced, particularly with the supplementation of folic acid as it is needed for the methylation of homocysteine into methionine, causing homocysteine`s levels to decrease [6]. Nonetheless, the clinical benefits of lowering homocysteine remain uncertain, as some studies have reported no significant reduction in CVD events despite decreased homocysteine levels. Currently, there is a gap in the literature, as most studies fail to solely study the importance of folic acid for cardiovascular disease prevention. In contrast, our study conducted a broader literature search across multiple databases and included subgroups and sensitivity analysis to provide a more comprehensive assessment. Although the latest meta-analysis by JAHA provided useful insights, it lacked a proper sensitivity analysis and focused solely on stroke, without updating data on cardiovascular disease, mortality, or congestive heart failure [7]. Some of our limitations are the different consensus between authors, the use of folic acid in conjunction with other medications, and the different levels of folic acid in participants due to their diet. In conjunction with other vitamin supplements, folic acid supplementation of 0.5–5.0.5.0 mg daily has been described to diminish homocysteine plasma levels by 25% [8].

Additionally, folic acid, by default, increases tetrahydrobiopterin (BH4) bioavailability, which notes importance in BH4’s implication in endothelial function since it is a cofactor of Nitric Oxide Synthase (NOS) enzyme, producing Nitric Oxide (NO) [9]. Thus, as a metabolism mediator for homocysteine and BH4, the role of folic acid in methionine metabolism may prove paramount to reducing CVD risk in the general population. Moreover, this systematic review and meta-analysis addressed the association of folic acid supplementation, which is a relatively low-cost intervention that may contribute to cardiovascular disease prevention. However, its cost-effectiveness compared to pharmacological therapies and lifestyle interventions has not been comprehensively evaluated.

Criteria for considering studies

Studies were eligible for inclusion if they met the following criteria: (1) enrolled adult participants aged 18 years or older; (2) evaluated the efficacy of orally administered folic acid supplementation, either as monotherapy or in combination with other interventions; and (3) reported at least one major cardiovascular clinical outcome, defined as cardiovascular disease (CVD), peripheral arterial disease (PAD), aortic dissection, congestive heart disease (CHD), or stroke, as well as cardiovascular risk markers, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), body mass index (BMI), or hemoglobin A1c (HbA1c). Studies were excluded if they: (1) included participants with known genetic, biochemical, or structural abnormalities affecting folate metabolism (e.g., MTHFR mutations, homocystinuria); (2) administered folic acid via parenteral routes; or (3) did not report extractable quantitative data for the outcomes of interest.

Methods

Search methods

Our search on April 3, 2025, encompassed Pubmed, Cochrane, Scopus, Web of Science, CINAHL and EMBASE using relevant MESH terms and free-text terms such as ‘’Folic Acid’’, ‘’heart disease’’, ‘’cardiovascular disease’’, this terms were adapted using Boolean operators and adapted to each database, for the entire search strategy refer to our supplementary material (Supplementary Tables 1–6). We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards to guide the systematic review articles selection process [10, 11]. Additionally, it was registered in PROSPERO under the registration number CRD42024525945.

Selection of studies

In the initial phase, titles and abstracts were screened by two independent reviewers (PEGH, JDMI), who selected studies for inclusion based on established criteria. Rayyan facilitated the search process, enabling the extraction of pertinent data and eliminating duplicate records [12]. Keywords were employed in Rayyan to pinpoint terms associated with inclusion and exclusion criteria (Supplementary material). Discrepancies between the two reviewers in study selection were harmonized through consulting a third reviewer (JAP). Subsequently, a detailed analysis of full texts was conducted, wherein two different reviewers (EP, BSSZ) independently assessed studies for inclusion, adhering to the predefined criteria. Conflicts concerning study selection were resolved by achieving consensus or through the mediation of a third reviewer (JAP).

Data extraction

Data extraction was conducted by two independent reviewers (MMGM and PEGH), using a standardized data extraction form developed in Microsoft Excel. The form was designed to capture key study characteristics and outcomes relevant to the review. Any discrepancies between reviewers were resolved through discussion and consensus. In cases where multiple overlapping reports from the same study were identified, we included the version that presented the most clearly reported data or provided a more comprehensive set of relevant variables. The extracted data included study design, participant age, sex, comorbidities, type, dose, and duration of the intervention, baseline and follow-up levels of folate and homocysteine, changes in homocysteine levels, and clinical outcomes related to cardiovascular disease, coronary heart disease, stroke, mortality, and peripheral arterial disease. Additional variables included LDL and HDL levels, among others.

Assessment of risk of bias in included studies

Our data evaluation adhered to the criteria established by the Cochrane Handbook [13]. For the assessment of study quality within our systematic review, the Cochrane Risk of Bias tool version 2.0 [14] was utilized for the evaluation of randomized controlled trials (RCTs) [14]. The tool assesses five specific domains of bias: (1) bias arising from the randomization process (2), bias due to deviations from intended interventions (3), bias due to missing outcome data (4), bias in measurement of the outcome, and (5) bias in selection of the reported result. Judgements were categorized as “low risk,” “some concerns,” or “high risk” of bias for each domain and overall. This process involved two independent reviewers (MMGM and ACAR) determining the risk of bias in each study based on the explicit criteria and guidelines provided by the tool. In instances of disagreement between reviewers, a resolution was achieved through consultation with a third reviewer, who remained blinded to the initial assessments (JLC). For the methodological evaluation of trials and case-control studies, the risk of bias was classified as low, high, or unclear per the directives in the Cochrane Handbook for Systematic Reviews of Interventions [13]. The rationale behind any decisions to downgrade or upgrade the quality of evidence will be elucidated in the summary of findings table, ensuring transparency and providing a detailed explanation for the bias assessment conducted for each study included in the review.

Statistical analysis

A meta-analysis was conducted using R version 3.4.3 (R Core Team). We examined the pooled effect of the outcomes using a random-effects meta-analysis based on the DerSimonian-Laird approach [15]. When the number of studies reporting an outcome of interest was insufficient, only a qualitative analysis of the results was performed. Effect sizes were expressed as relative risk (RR) or standardized mean difference (SMD), along with their corresponding 95% confidence intervals. The I2 statistic was employed to assess heterogeneity, with the following cut-off values used for interpretation: <25, 25–50, and >50% were considered small, medium, and large heterogeneity respectively [16]. Sensitivity analyses were conducted for all outcomes using the leave-one-out method to determine the influence of individual studies on the overall effect, studies were evaluated using standard influence measures—such as studentized residuals, DFFITS, Cook’s distance, covariance ratio, and hat values. Studies exceeding conventional thresholds (e.g., standardized residuals >2 or hat values >3p/n) were flagged as influential [17]. To assess publication bias, funnel plots were visually inspected for asymmetry, in addition Egger’s regression test was used to assess publication bias when 10 or more reports with the same outcome were available [18]. Additionally, subgroup analyses were performed for primary outcomes, considering variables such as year, country, sex, risk of bias, folate dose, previous cardiovascular history, renal disease, history of stroke or diabetes, duration and type of intervention, mean age, baseline folate levels, and changes in homocysteine levels, among others. The subgroup approach aimed to explore potential sources of heterogeneity rather than to infer causal relationships, and findings should be interpreted accordingly.

Results

The methodology for identifying and selecting pertinent studies involved a comprehensive search across multiple databases, yielding a preliminary list of 25,881 articles. Subsequent evaluation facilitated the removal of 9,454 duplicate articles. Screening of titles and abstracts further narrowed down the selection to 69 reports for retrieval, of which 8 were not obtained. Additionally, 30 records were identified from a previous meta-analysis, but only 27 were retrieved. After assessing the full texts of the remaining reports from the databases, including the ones in the previous study, 16 were excluded due to wrong study design, outcome, or population. Ultimately, 45 studies were included in the final review, as depicted in the PRISMA flow diagram in Fig. 1 [19]. The process of screenings reached a Cohen’s Kappa of 0.95 between reviewers [20].

Fig. 1.

PRISMA flow diagram. Flow diagram illustrating the study selection process for the systematic review. The initial search across six databases yielded a total of 25,881 records. After removing 9,454 duplicate records, 16,427 titles and abstracts were screened for relevance. Following the screening, 69 full-text reports were sought for detailed evaluation. Eight of these could not be retrieved. Of the 61 reports assessed for eligibility, 16 were excluded based on specific exclusion criteria, including wrong study design (n = 11), wrong outcome (n = 1), and wrong population (n = 4). A remaining of 45 studies met the inclusion criteria and were incorporated into the systematic review. From these articles, 18 new articles were added in comparison to the previous study. This process adheres to the PRISMA guidelines for reporting systematic reviews and meta-analyses

In evaluating the risk of bias for the 45 studies under consideration, the outcomes, shown in Fig. 2, indicate that the majority of studies exhibited a low risk of bias across most domains. Specifically, a low risk of bias was observed in over 75% of studies in categories such as randomization process, deviations from intended interventions, missing outcome data, and measurement of outcomes. However, some concerns were identified in the selection of reported results, with a small proportion of studies also raising concerns in the randomization process, and deviations from intended interventions. A minority of studies demonstrated a high risk of bias, particularly in the domains of missing outcome data, measurement of outcomes, and the selection of reported results. Overall, this analysis, visualized in Fig. 2, highlights that while most studies had a low risk of bias, certain areas, such as outcome measurement and selective reporting, require cautious interpretation.

Fig. 2.

ROB. This represents a risk of bias summary table evaluating 45 studies across five bias domains (D1-D5). Domains include randomization, intervention deviations, missing data, outcome measurements, and reported result selection. The color-coded system: green for low risk, yellow for some concerns, and red for high risk, visually conveys the risk in each domain and overall, per study

The systematic review focuses on evaluating the effects of folic acid therapy on cardiovascular outcomes across various populations and conditions. The studies included in our analysis span a broad geographic range, reflecting diverse populations and a wide spectrum of cardiovascular conditions. In total, the analysis included 96,962 participants. Notably, China contributed the largest proportion of participants, accounting for 24.96% of the total sample. The United States also had a significant share with 15.41%, followed by the United Kingdom at 12.86%, and Canada at 12.55%. Australia contributed 8.74%, and the Netherlands accounted for 6.03% of the total sample. Norway comprised 5.34%, while Brazil added 4.43% and Scotland contributed 3.80%. Other countries, though contributing smaller percentages, add to the global diversity of our research: France (2.58%), Germany (1.33%), Switzerland (0.57%), New Zealand (0.32%), India (0.31%), Italy (0.25%), the Philippines (0.25%), Iran (0.10%), Egypt (0.10%), and Sweden (0.06%). This wide geographical distribution ensures a comprehensive understanding of the impact of folic acid supplementation on cardiovascular health across different populations and environmental conditions.

The compiled evidence addresses cardiovascular health in various clinical scenarios, ranging from stable coronary artery disease to chronic renal failure. Most studies employed randomized controlled trial designs, focusing on folic acid’s potential to modulate homocysteine levels, an established risk factor for cardiovascular disease. While results varied, significant benefits were observed in several studies, including reductions in homocysteine levels and improvements in vascular function. However, some studies reported no clear advantage of folic acid therapy over placebo in preventing cardiovascular events. This heterogeneity underscores the complexity of cardiovascular disease mechanisms and highlights the necessity for future research to clarify folic acid’s role within the broader context of cardiovascular risk management. Detailed findings and study characteristics such total sample size, duration of treatment, baseline folate levels, homocysteine levels, if given with Vitamin B, and Folic acid dose given are encapsulated in Table 1 [21–65].

Table 1.

General outcome table

Author, Year, Country	Sex	Total Sample Size	Duration Months	Folate Baseline	Homocysteine			FA Plus Other Vit B	FA dose mg/day	Key points
					Baseline	Change	Change %
Mehta K, 2005 [21], India	Both	124	3	NA	NA	NA	NA	No	5	84 patients with documented CAD and 40 control subjects, primary outcome was reduction in t-HCY levels after folate therapy in patients with hyperhomocysteinemia, they found a significant reduction in t-HCY levels in patients with mild and moderate hyperhomocysteinemia but nts with severe no reduction in patients with severe
Zoungas S, 2006 [22], Australia/New Zealand	Both	315	42	NA	27	−4.7	−17.4	No	15	156 patients with ESRD and 159 healthy controls were recruited, the aim of the study was to establish whether high-dose folic acid would slow the progression of atherosclerosis and reduce cardiovascular events in patients with chronic renal failure (CRF), acid therapy was shown to not slow atherosclerosis folic acid therapy was shown to not slow atherogression or improve cardiovascular morbidity or mortality in patients with CRF.
Bleie O, 2011 [23], Norway	Both	40	24	12.2	10.7	−3.3	−30.8	Yes	0.8	40 patients with stable CAD were randomly assigned to folic acid + VB12 therapy or placebo, the objective of the study was to assess the effect of folic acid + VB12 on coronary vascular function, they found a significant increase in basal and adenosine-induced coronary blood flow in the folic acid + VB12 group.
Liu M, 2020 [24], China	Both	99	3	NA	13.1	−1.47	−11.2	Yes	0.4	42 patients with CAD and 48 healthy subjects were studied, the objective of the study was to evaluate the effect of B vitamins supplementation on dyslipidemia and inflammatory cytokines, they found improvement in hcy levels, TG,TC, HDL-C, IL-10 and IL-1B. CC,
Shidfar F, 2009 [25], Iran	Both	40	2	5.08	12.91	−3.98	−30.8	No	5	40 patients with hypercholesterolemia were randomly assigned to folate supplementation and placebo therapy for 8 weeks, the aim of this study was to assess the effect of folate therapy on hcy levels and total antioxidant capacity, they found a significant decrease in HCY levels and a significant increase in total antioxidant capacity in folate group.
Schnyder G, 2002 [26], Switzerland	Both	553	11	NA	11.2	−2.9	−25.7	Yes	1	553 patients with h/o successful angioplasty of at least 1 significant coronary stenosis were randomly assigned to a Folic acid + Vit. B12 + Vit. B6 or placebo therapy for 6 months. Follow up of 11 months resulted on significantly reduced rate of target lesion revascularization, decreasing the incidence of major adverse events after percutaneous coronary intervention.
Vermeulen E, 2000 [27], Netherlands	Both	158	24	12.1	14.7	−4.6	−31.3	Yes	5	158 Healthy siblings (siblings of 167 patients with h/o premature atherothrombotic disease) were assigned to Placebo or FA + Vit. B6 daily for 24 months (about 2 years). The results showed lowering of fasting HCY concentration and decreased risk of atherosclerotic coronary events
VITATOPS Trial, 2010 [28], Australia	Both	8164	41	NA	14.3	−4	−30	Yes	2	8164 patients with history of recent stroke or TIA were randomly assigned to daily administration of FA + Vit. B6 + Vit. B12 or placebo. Results showed that B vitamins were safe but were not more effective than placebo in reducing major vascular events.
Van Dijk S, 2015 [29], Netherlands	Both	2919	24	18.7	14.4	−3.8	−26.4	Yes	0.4	2919 patients were randomly assigned to FA + Vitamin B12 or placebo, compared to placebo b vitamin supplementation lowered serum homocysteine, but had no effect on PWV levels, or carotid IMT.
Doshi S, 2002 [30], UK	Both	33	1.5	22.37	10.7	−2.3	−21.5	No	5	33 patients were randomly assigned to FA or placebo, after assessing the flow-mediated dilatation FMD. Data collected at 6 weeks (about 1 and a half months) showed that FA improves endothelial function in CAD
Bonaa K, 2006 [31], Norway	Both	2815	36	12.4	13.1	−3.8	−27.5	Yes	0.8	Patients were divided into groups of folic acid with vitamins B and placebo. Folic acid + B12, with or without vitamin B6, did not significantly reduce the risk of the primary outcome (MI, stroke, sudden death;risk ratio, 1.08; 95% confidence interval, 0.93 to 1.25; P = 0.31), as compared with placebo. Folic acid + B12 + B6 group, showed increased risk in primary outcome (relative risk, 1.22; 95% confidence interval, 1.00 to 1.50; P = 0.05), so it is not recommended by the study.
Toole J, 2004 [32], United States/Canada/Scotland	Women	3680	24	26	13.4	−2.3	−17.2	Yes	2.5	Mean reduction of total homocysteine was 2 µmol/L greater in the high- dose group than in the low-dose group, but there was no treatment effect on any end point. The unadjusted risk ratio for any stroke, CHD event, or death was 1.0 (95% confidence interval [CI], 0.8–1.1), with chances of an event within 2 years of 18.0% in the high-dose group and 18.6% in the low-dose group. The risk of ischemic stroke within 2 years was 9.2% for the high-dose and 8.8% for the low-dose groups (risk ratio, 1.0; 95% CI, 0.8–1.3;P = 0.8)0 by log-rank test of the primary hypothesis of difference in ischemic stroke between treatment groups).
Guo H, 2009 [33], China	Both	82	2	NA	NA	NA	NA	No	5	The plasma Hcy levels of 52 cases with UA and 30 control subjects were measured by using high-performance liquid chromatography (HPLC) with fluorescence detection, plasma folic acid and vitamin B12 levels were also measured.
Jonasson T, 2005 [33], Sweden	Both	60	3	NA	17.5	−10.5	−60	Yes	5	60 patients randomized into 5 mg folic acid + pyridoxine + cyanocoobalamin and placebo. Folic acid therapy reduced plasma tHCY from 17.4 to 9.2 mmol/L (p-0.0001).
Rydlewicz A, 2001 [35], UK	Both	368	1.5	NA	10.54	−2.06	−19.54	No	0.4	Only the 400 µg and 600 µg groups had significantly lower homocysteine levels compared to placebo (p = 0.038 and p < 0.001, respectively).Using multiple linear regression and each individual’s total folic acid intake (diet plus supplement), a total daily folic acid intake of 926 µg per day would be required to ensure that 95% of the elderly population would be without cardiovascular risk from folate deficiency.
Huo Y, 2015 [35], China	Both	20,702	54	8.1	12.5	NA	NA	No	0.8	During a median treatment duration of 4.5years, compared with the enalapril alone group, the enalapril-folic acid group had a significant risk reduction in first stroke (2.7% of participants in the enalapril–folic acid group vs. 3.4% in the enalapril alone group; hazard ratio [HR], 0.79; 95% CI, 0.68–0.93), first ischemic stroke (2.2% with enalapril–folic acid vs. 2.8% with enalapril alone; HR, 0.76; 95% CI, 0.64–0.91), and composite cardiovascular events consisting of cardiovascular death, MI, and stroke (3.1% with enalapril–folic acid vs. 3.9% with enalapril alone; HR, 0.80; 95% CI, 0.69–0.92).
Lange H, 2004 [37], Germany/Netherlands	Both	636	6	NA	12.6	−3.6	−28.6	Yes	1.2	636 participants with history of coronary stent received 1.2 mg folic acid + B6 and B12 or placebo. Folate therapy increased risk of restenosis compared to placebo group (34.5% vs. 26.5%, P = 0.05), and a higher percentage required repeated target-vessel revascularization (15.8% vs. 10.6%, P = 0.05).
Liem A, 2004 [37], Netherlands	Both	283	12	NA	NA	NA	NA	No	5	At 12 months, lipid parameters were similar for both groups; mean total cholesterol value was 5.5 mmol/l (S.D. 0.9) in the folic acid group and 5.7 mmol/l (S.D. 0.9) in the control group. Mean LDL values were 3.3 mmol/l (S.D. 0.8) and 3.4 mmol/l (S.D. 0.8), respectively. No significant changes in mean HDL or triglycerides were observed In this study, administration of folic acid, when added to statin therapy, did not have any beneficial effects on cardiovascular mortality or morbidity in a population of post-AMI patients with a relative high cholesterol value at admission
Wrone E, 2004 [38], United States	Both	528	24	47.16	32.9	−3.6	−10.9	Yes	5	510 patients on chronic dialysis were randomized to 1, 5, or 15 mg of folic acid contained in a renal multivitamin. Mortality and cardiovascular events did not vary (43.7% in 1 mg group, 38.6% in 5 mg group, 47.1% in 15 mg group; lP = 0.47) Folic acid supplementation in ESRD is unlikely to produce benefit at doses between 1 and 15 mg/d.
Liem A, 2003 [40], Netherlands	Both	593	24	17	12.1	−2.6	−21.4	No	0.5	593 patients on statins for a mean of 3.2 years were randomized into folic acid (n = 300) or placebo (n = 293). In folic acid group homocysteine levels decreased by 18%, from whilecontrol group was unaffected (p 0.001 between groups). The primary end point (all-cause mortality and a composite of vascular events) was seen in 31 (10.3%) patients in the folic acid group and in 28 (9.6%) patients in the control group (relative risk 1.05; 95% confidence interval: 0.63 to 1.75).
Liem A, 2005 [41], Netherlands	Both	593	42	16	12.1	−2.6	−21.5	No	0.5	There were no significant differences between the groups (log rank test, p = 0.53); the relative risk for total events was 0.85 (95% confidence interval 0.56 to 1.31).
Lonn E, 2006 [41], Canada	Both	5522	60	27.4	NA	−3.2	−26.2	Yes	2.5	Mean plasma homocysteine levels decreased by 2.4 µmol per liter (0.3 mg per liter) in the active-treatment group and increased by 0.8 µmol per liter (0.1 mg per liter) in the placebo group. Supplements combining folic acid and vitamins B6 and B12 did not reduce the risk of major cardiovascular events in patients with vascular disease.
Righetti M, 2006 [43], Italy	Both	88	28	6.8	37.2	−15.1	−43.6	Yes	5	Homocysteine-lowering folate therapy decreases cardiovascular events in dialysis patients. It is necessary to perform large prospective studies to confirm results.
Jamison R, 2007 [43], United States	Both	2056	38	15.6	24	−5.9	−24.5	Yes	40	Treatment with high doses of folic acid and B vitamins did not improve survival or reduce the incidence of vascular disease in patients with advanced chronic kidney disease or end-stage renal disease.
Vianna A, 2007 [45], Brazil	Both	186	24	10	23.5	−13	−55.3	No	4.29	Folate treatment for only 2 years was not effective in modifying cardiovascular death and nonfatal cardiovascular events in this sample of patients with chronic uremia
Ebbing M, 2008 [45], Norway	Both	2324	38	NA	10.8	−3.2	−29.6	Yes	0.8	The article examines the effects of B-vitamin supplementation on homocysteine levels and its potential to reduce cardiovascular risk in 3096 adult participants undergoing coronary angiography. The study found no significant benefit in reducing mortality or cardiovascular events despite lowering homocysteine levels.
Severino M, 2009 [46], Philippines	Both	243	6	NA	12.8	NA	NA	Yes	1	The study comprised 240 patients with either unstable angina or non-ST-elevation myocardial infarction in the previous 2 weeks who were randomized to a folate group (n = 116) or a placebo group (n = 124). The composite endpoint of death, nonfatal acute coronary syndrome, and serious re-hospitalization was significantly higher in the folate group; serious re-hospitalization alone was significantly higher in this group. Folic acid-based supplementation is not beneficial and may even be harmful.
Armitage J, 2010 [48], UK	Both	12,064	80	16.8	13.5	−3.8	−28	Yes	2	Double-blind randomized controlled trial of 12,064 survivors of myocardial infarction. There were no apparent effects on major coronary events (vitamins, 1229 [20.4%], vs. placebo, 1185 [19.6%]; RR, 1.05; 95% CI, 0.97–1.13), stroke (vitamins, 269 [4.5%], vs. placebo. Substantial long-term reductions in blood homocysteine levels with folic acid and vitamin B(12) supplementation did not have beneficial effects on vascular outcomes
Heinz J, 2010 [49], Germany	Both	650	24	14.3	29	−8.6	−30	Yes	5	Randomly assigned 650 patients with end-stage renal disease. Increased intake of folic acid, vitamin B(12), and vitamin B(6) did not reduce total mortality and had no significant effect on the risk of cardiovascular events in patients with end-stage renal disease.
House A, 2010 [50], Canada	Both	238	32	16	15.5	−4.8	−31	Yes	2.5	B-vitamin therapy (folic acid, B6, B12) resulted in a greater decrease in glomerular filtration rate (GFR) over 36 months in the treatment group compared to placebo, indicating faster progression of nephropathy in those receiving B vitamins. The B-vitamin group also experienced an increased risk of vascular events (hazard ratio: 2.0), suggesting that high-dose supplementation may worsen vascular outcomes in diabetic nephropathy patients.
Bostom A, 2011 [51], United States/Canada/Brazil	Both	4110	48	NA	16.4	−4.4	−26.8	Yes	5	High-dose folic acid, vitamin B6, and B12 supplementation significantly reduced homocysteine levels, but this did not lead to a reduction in cardiovascular disease events or all-cause mortality in kidney transplant recipients. The study found no significant difference between the high-dose and low-dose multivitamin groups for the composite cardiovascular outcome, despite the homocysteine-lowering effect.
Righetti M, 2003 [52], Italy	Both	81	12	5.98	50.3	−26	−51.7	No	5	Folic acid treatment reduced homocysteine levels in hemodialysis patients, but 88% of treated patients maintained higher-than-normal levels of homocysteine. A trend was observed towards reduced cardiovascular events in the treated group, but this reduction was not statistically significant.
Nand N, 2013 [53], India	Both	100	6	11.93	31.06	−17.84	−57.44	Yes	2.5	Folic acid and vitamin B12 supplementation significantly reduced homocysteine levels in patients with chronic kidney disease (CKD) after 6 months (from 32.61 µmol/L to 19.69 µmol/L in the treatment group). Despite the reduction in homocysteine levels, supplementation did not lead to a significant decrease in cardiovascular disease mortality or other cardiovascular outcomes.
Bahmani F, 2018 [54], Iran	Women	60	3	NA	19.9	−3.5	−17.6	No	5	The study focused on a sample of 60 women diagnosed with endometrial hyperplasia (EH) for 3 months. The research examined the effects of folic acid supplementation at a daily dose of 5 mg, although no additional vitamin B12 was provided. After the intervention, folic acid supplementation significantly improved glycemic control, triglycerides, VLDL, cholesterol, and hs-CRP levels , but did not influence recurrence or other metabolic profiles.
Maurizi A, 2016 [55], Italy	Both	26	6	NA	NA	NA	NA	Yes/no	0,4	The study focused on a sample of 26 patients with type 1 diabetes who were overweight or obese, undergoing intensive insulin therapy for 6 months. The research examined the effects of folic acid supplementation at a daily dose of 0.4 mg, although no additional vitamin B12 but it was provided additional D-chiroinositol (DCI). After 3-month follow-up, a significant reduction in HbA1c levels was observed in DCI-treated group versus control group [7.5% (58 mmol/mol) ± 1.1 vs. 8.1% (65 mmol/mol) ± 1.9, respectively, p\0.05]. At the end of the study period (6 months), HbA1c reduction in DCI treated group vs. control group was statistically confirmed.
El-khodary N, 2022 [56], Egypt	Both	100	3	NA	1,7	−0,4	23,5	No	5	This clinical trial has been performed on 100 patients with T2DM randomly folic acid 5 mg/d for 3 months. although no additional vitamin B12 was provided. After the intervention, folic acid supplementation caused a significant decrease in homocysteine and sortilin serum levels (28.2% and 33.7%, P < 0.0001, respectively). After 3 months of intervention, 8.7% decrease in fasting blood glucose (P = 0.0005), 8.2% in HbA1c (P = 0.0002), 13.7% in serum insulin (P < 0.0001) and 21.7% in insulin resistance (P < 0.0001) were found in the folic acid group.
Satapathy S, 2020 [57], India	Both	80	2	NA	4,9	−2,45	−50	Yes	5	80 patients with type 2 diabetes and oral antidiabetics were divided on 4 groups: folic acid, Methylcobalamin, Folic acid + methylcobalamin, or antidiabetic alone. Methylcobalamin and Folic acid + methylcobalamin groups showed improvement in HbA1c, plasma insulin, insulin resistance and serum adiponectin compared to antidiabetic alone. Homocysteine levels improved in all groups except antidiabetics alone, and there was no lipids improvement in any of the groups.
Araghi S, 2020 [58], Netherlands	Both	1298	54	21.4	14.4	−0.1	−0.69	Yes	0.4	1,298 individuals (44.5%) participated in the second follow-up round, with a median follow-up of 54 months. The intervention with folic acid and vitamin B12 showed no significant effect on osteoporotic fracture risk or any fracture risk (HR: 0.99 and HR: 0.77, respectively). However, a potential interaction was noted with baseline homocysteine concentrations, indicating a lower risk of any fracture in the treatment group with higher homocysteine levels (> 15.1 mmol/l). No age-dependent effects were found.
Albert CM, 2008 [59], United States	Women	5442	88	NA	12.3	−2.3	−18.5	Yes	2.5	This study tested whether a combination of folic acid, vitamin B6, and vitamin B12 reduces cardiovascular disease (CVD) risk among high-risk women. Involving 5,442 female US health professionals, the trial followed participants for 88 months. Results showed that the treatment did not significantly reduce the risk of myocardial infarction, stroke, coronary revascularization, or CVD mortality compared to placebo, despite lowering homocysteine levels. The conclusion is that this vitamin combination does not reduce total cardiovascular events in high-risk women.
Cole B, 2007 [60], United States/Canada	Both	1021	84	23.8	9.8	NA	NA	No	1	1021 participants were involved in an 84-month study that assessed folic acid’s effectiveness in preventing colorectal adenomas. It found no significant difference in adenoma incidence between the folic acid and placebo groups. Furthermore, folic acid was linked to higher risks of multiple adenomas and non-colorectal cancers leading to the conclusion that folic acid does not reduce colorectal adenoma risk and may increase colorectal neoplasia risk.
Mark S, 1996 [61], China	Both	3318	72	NA	NA	NA	NA	Multi-vitaminicuco	0.8	3,318 participants were part of an RCT study that evaluated the effects of a multivitamin/mineral supplement on cancer and cerebrovascular disease. Over 72 months, the supplement group showed a slight reduction in overall mortality (RR = 0.93) and cerebrovascular disease deaths (RR = 0.63), particularly among men. Blood pressure elevations were less common in the supplement group. The findings suggest that such supplements may reduce mortality from cerebrovascular disease and hypertension in populations with micronutrient-poor diets.
Potena L, 2008 [62], Italy	Both	51	12	NA	17.9	NA	NA	No	15	51 participants after heart transplantation were randomized into 15 mg/day of methyltetrahydrofolate or standard therapy were followed up for 7-year outcome. Survival was greater in recipients randomized to folate (88%± 6% vs. 61%±9%, P = 0.04) as well as lower mortality (relative risk [RR] 0.53, 95% confidence interval [CI] 0.25–0.97; P = 0.036), Also, decreased mortality was seen in High-risk subgroup which included participants >55 years old and patients transplanted because of coronary artery disease (RR 0.43, 95% CI 0.17–0.85) but not in the lower-risk subgroup (RR 1.11, 95% CI 0.22–5.61).
Hodis H, 2009 [63], United States	Both	506	37	21.4	9.7	−2.1	−21.6	Yes	5	506 participants with initial tHcy ≥ 8.5 mol/L without diabetes and cardiovascular disease were randomized to high-dose B vitamin supplementation (5 mg folic acid + 0.4 mg vitamin B12 + 50 mg vitamin B6) or placebo for 3.1 years. In participants with baseline tHcy ≥ 9.1 mol/L, those randomized to B vitamin supplementation had a statistically significant lower average rate of carotid artery intima media thickness progression compared with placebo (P = 0.02)
Lamas G, 2013 [64], United States/Canda	Both	1708	55	NA	NA	NA	NA	Yes	0.8	1708 patients aged ≥ 50 years who had MI at least 6 weeks earlier and serum creatinine levels of 176.8 mol/L (2.0 mg/dL) or less, were randomized into high-dose multivitamin and multiminerals or placebo. High-dose multivitamins group did not statistically significantly reduce cardiovascular events in patients after MI who received standard medications. Cardiovascular death, MI, or stroke occurred in 94 (11%) patients in the vitamin group and 115 (13%) in the placebo group (hazard ratio, 0.82 [CI, 0.62 to 1.07]; P 0.142).
Galan P, 2010 [65], France	Both	2501	56	15.2	12.8	−2.9	−22.7	Yes	0.56	2501 patients with a history of myocardial infarction, unstable angina, or ischaemic stroke were randomized into dietary supplement containing 5-methyltetrahydrofolate (560 µg), vitamin B-6 (3 mg), and vitamin B-12 (20 µg, omega 3 fatty acid or placebo. B vitamins group lowered plasma homocysteine concentrations by 19% compared with placebo, but had no significant effects on major vascular events (75 vs. 82 patients, hazard ratio, 0.90 [95% confidence interval 0.66 to 1.23, P = 0.50]). Study does not support the routine use of dietary supplements containing B vitamins or omega 3 fatty acids for prevention of cardiovascular disease in patients with a history cardiovascular disease.

RCT Randomized control trial, NA Not applicable, CAD Coronary artery disease, t-HCY Total homocysteine, HCY Homocysteine, ESRD End-stage renal disease, CRF Chronic renal failure, VB12 Vitamin B-12, TG Triglycerides, TC Total cholesterol, LDL-C Low-density lipoprotein cholesterol, HDL-C High-density lipoprotein cholesterol, IL-10 Interleukin 10, IL-1B Interleukin 1B, H/O History of, Vit. B12 Vitamin B12, Vit. B6 Vitamin B6, FA Folic acid, HX History, TIA Transient ischemic attack, PWV Pulse wave velocity, IMT Intima-media thickness, FMD Flow-mediated dilation, CHD Coronary heart disease, UA Unstable angina, HPLC High-performance liquid chromatography, mg Milligrams, g Grams, MI Myocardial infarction, HR Heart rate

Meta-analysis

A meta-analysis was conducted to assess the relationship between folic acid supplementation and the risk of cardiovascular disease, stroke, coronary heart disease, peripheral arterial disease, and mortality. Additionally, the influence of folic acid supplementation on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels was evaluated.

Overall, folic acid supplementation was associated with a statistically significant reduction in stroke risk (RR = 0.85, 95% CI: 0.76; 0.96, p < 0.01) and a small but statistically significant reduction in cardiovascular disease (RR = 0.95, 95% CI: 0.90 to 0.99, p = 0.04). No significant risk reduction was observed for mortality, coronary heart disease, or peripheral arterial disease. Additionally, folic acid supplementation did not result in statistically significant changes in HDL or LDL levels across studies. It is important to note that some analyses showed moderate heterogeneity, for which sensitivity analyses were performed to account for it and to investigate potential publication bias.

Cardiovascular disease

We analyzed 25 studies focusing on cardiovascular disease risk, which included a total of 79,589 individuals. Using a random-effects model, patients receiving folic acid supplementation had a RR of 0.95 (95% CI: 0.90 to 0.99, p = 0.04, I² = 21%). The prediction interval ranged from 0.84 to 1.07 (Fig. 3A).

Fig. 3.

Effect of folic acid supplementation on cardiovascular disease risk. A Forest plot detailing the relative risk (RR) and 95% confidence intervals for the effect of folic acid supplementation on cardiovascular disease risk. B Funnel plot detailing publication bias in the included studies

Funnel plot in cardiovascular disease risk

The funnel plot appeared asymmetrical, suggesting publication bias, though Egger’s test did not show statistical evidence of publication bias (p = 0.14; Fig. 3B).

Subgroup and sensitivity analysis

Subgroup analyses were performed to explore differences among intervention groups (p = 0.57) and homocysteine changes (p < 0.01). The group receiving folic acid alone showed statistically significant results with no heterogeneity (RR: 0.82, 95% CI: 0.73 to 0.91, I²=0%). Similarly, the group with Hcy > 4 micromol/L showed a significant effect (RR:0.90, 95% CI: 0.84 to 0.96, I2 = 0%). Other subgroups did not show statistically significant differences (Supplementary Table 7). Sensitivity analysis identified two studies, Armitage (2010) and Huo (2015), as potential outliers. Excluding these articles reduced heterogeneity without affecting the effect size (RR: 0.95, 95% CI: 0.91 to 0.99, I2 = 0%; Supplementary Table 8).

Stroke

We analyzed 23 studies focusing on stroke risk, including 83,247 individuals. Using the random-effects model, patients receiving folic acid supplementation had a stroke risk of RR 0.85 (95% CI: 0.76; 0.96, p < 0.01, I2 = 47%). The prediction interval ranged from 0.59 to 1.23 (Fig. 4A).

Fig. 4.

Effect of folic acid supplementation on stroke risk. A Forest plot detailing the relative risk (RR) and 95% confidence intervals for the effect of folic acid supplementation on stroke risk. B Funnel plot detailing publication bias in the included studies.

Funnel plot in stroke

The funnel plot appeared asymmetrical, though Egger’s test did not show statistical evidence of publication bias (p = 0.47) (Fig. 4B).

Subgroup and sensitivity analysis

Subgroup analyses showed differences among placebo groups (p = 0.04). The group receiving a regular placebo, instead of a lower-dose folic acid placebo, showed significant effects (RR: 0.81, 95% CI: 0.71 to 0.93, I²=51.3%). Studies including both sexes also had significant effects (RR: 0.82, 95% CI: 0.72 to 0.92, I2 = 43.9%). Other subgroup analyses did not show statistically significant differences (Supplementary Table 9). Sensitivity analysis identified Ebbing (2008) as a potential outlier. Excluding this study reduced heterogeneity without affecting the effect size (RR: 0.89, 95% CI: 0.82 to 0.97, I2 = 24%; Supplementary Table 10).

Coronary heart disease

We analyzed 22 studies focusing on coronary heart disease, which included 80,923 individuals. Using the random-effects model, patients with folic acid supplementation had a myocardial infarction risk of RR 0.98 (95% CI: 0.91 to 1.06, p = 0.64, I2 = 26%). The prediction interval ranged from 0.77 to 1.24 (Fig. 5A).

Fig. 5.

Effect of folic acid supplementation on coronary heart disease risk. A Forest plot detailing the relative risk (RR) and 95% confidence intervals for the effect of folic acid supplementation on coronary heart disease risk. B Funnel plot detailing publication bias in the included studies.

Funnel plot in coronary heart disease

The funnel plot appeared asymmetrical, though Egger’s test did not show statistical evidence of publication bias (p = 0.35; Fig. 5B).

Subgroup and sensitivity analysis

Subgroup analyses did not reveal significant differences among groups (Supplementary Table 11). Sensitivity analysis identified Armitage (2010) and Ebbing (2008) as potential outliers. Excluding these studies reduced heterogeneity without affecting the effect size (RR: 1.01, 95% CI: 0.95 to 1.08, I2 = 0%; Supplementary Table 12).

Mortality

We analyzed 25 studies focusing on mortality risk, including 80,505 individuals. Using the random-effects model, patients with folic acid supplementation had a mortality risk of RR 0.98 (95% CI: 0.93 to 1.02, p = 0.25, I2 = 11%). The prediction interval ranged from 0.92 to 1.03 (Fig. 6A).

Fig. 6.

Effect of folic acid supplementation on mortality risk. A Forest plot detailing the relative risk (RR) and 95% confidence intervals for the effect of folic acid supplementation on mortality risk. B Funnel plot detailing publication bias in the included studies

Funnel plot asymmetry in mortality

The funnel plot appeared asymmetrical, though Egger’s test did not show statistical evidence of publication bias (p = 0.29; Fig. 6B).

Subgroup and sensitivity analysis

Subgroup analyses did not reveal significant differences among groups (Supplementary Table 13). Sensitivity analysis identified VITATOPS Trial (2010), Jamison (2007), and Armitage (2010) as potential outliers. Excluding these studies reduced heterogeneity and affected the effect size (RR:0.93, 95% CI: 0.87 to 0.99, I2 = 11%; Supplementary Table 14).

Peripheral arterial disease

We analyzed 4 studies on peripheral arterial disease, which included 5,233 individuals. Using the random-effects model, the risk of peripheral arterial disease in patients with folic acid supplementation was RR 0.94 (95% CI: 0.75 to 1.17, p = 0.58, I2 = 0%). The prediction interval ranged from 0.77 to 1.24 (Supplementary Fig. 1). Due to the low number of studies, it was not possible to generate a funnel plot. Subgroup and sensitivity analysis were not performed due to low heterogeneity.

High-density lipoprotein (HDL) levels after intervention

We analyzed 7 studies focusing on HDL levels after folic acid supplementation, including 21,675 individuals. Using the random-effects model, the mean difference (MD) in HDL levels was 0.31 (95% CI: −0.04; 0.65, p = 0.13, I2 = 39%). The prediction interval ranged from − 1.56 to 3.03 (Supplementary Fig. 2). Due to the low number of studies, it was not possible to generate a funnel plot.

Subgroup and sensitivity analysis

Subgroup analysis revealed statistical differences in the intervention subgroups (p = 0.03), though none of the subgroups showed statistically significant results (Supplementary Table 15). Sensitivity analysis identified El-khodary (2022), Huo (2015), Lange (2004), and Shidfar (2009) as potential outliers. Excluding these studies reduced heterogeneity and affected the effect size (RR: −0.59, 95% CI: −4.09 to 2.92, I2 = 0%; Supplementary Table 16).

Low-density lipoprotein (LDL)

We analyzed 6 studies focusing on LDL levels after folic acid supplementation, including 973 individuals. Using the random-effects model, the effect was MD −1.59 (95% CI: −8.82 to 5.64, p = 0.66, I2 = 63%). The prediction interval ranged from − 23.71 to 20.5 (Supplementary Fig. 3). Due to the low number of studies, it was not possible to generate a funnel plot.

Subgroup and sensitivity analysis

Subgroup analysis showed significant differences by year, country, and intervention (Supplementary Table 17). Sensitivity analysis identified Liu (2020) as an outlier. Excluding this study reduced heterogeneity without affecting the effect size (RR: 1.18, 95% CI: −2.87 to 5.23, I2 = 0%; Supplementary Table 18).

Discussion

Our meta-analysis was based on 45 RCTs and 96,962 participants with geographical diversity, predominantly from China, the United States, the United Kingdom, and Canada. Compared to the most recent meta-analysis of folic acid supplementation in CVD by Li et al. (2016), who included 30 RCTs and 82,334 participants [7]. Both studies conducted stratified analysis and included RCTs without restriction in sample size, treatment period, or preexisting diseases. In contrast, in our study, when the RCT assessed different doses of folic acid supplementation, we took into consideration the largest sample size group as cases and vitamins alone or placebo as controls for evaluating the levels of homocysteine [30, 34, 38, 45, 51, 56]. Due to the reasons above mentioned, a small variation of the results was shown, like a higher percentage of prevention in stroke risk, while no notable improvement was found overall in the placebo subgroup.

Compared with previous meta-analyses, our study incorporates several methodological enhancements. While Yang et al. (2012) searched three databases (PubMed, EMBASE, Cochrane Library) and Li et al. (2016) searched four (PubMed, EMBASE, Cochrane, and clinical trial registries), we broadened our search to six major databases (PubMed, Cochrane, Scopus, Web of Science, CINAHL, EMBASE), increasing comprehensiveness and population diversity [7, 66]. Both of them provided only limited information on baseline folate or homocysteine levels, whereas our study included comprehensive participant and intervention data covering comorbidities, folate/homocysteine concentrations, dosing, and intervention duration, allowing for more detailed subgroup and sensitivity analyses. Methodologically, Yang primarily used fixed-effects models and Li a mix of fixed- and random-effects models without extensive heterogeneity exploration; in contrast, we applied DerSimonian–Laird random-effects models with influence diagnostics, leave-one-out analyses, and stratification by dose, baseline folate status, and comorbidities. Our risk-of-bias assessment followed the updated Cochrane Risk of Bias 2.0 tool with dual independent reviewers, exceeding the single-assessor or older RoB tools used in earlier studies. Finally, our work was prospectively registered in PROSPERO and adhered strictly to PRISMA reporting standards, improving transparency and reproducibility compared with Yang (no registration reported) and Li (registration unclear) [7, 66].

Regarding the results of stroke outcome, the previous meta-analysis from Li et al. (2016) reported (RR = 0.90, 95% CI 0.84 to 0.96, p = 0.002) and Zhang N (2024) (RR 0.90, 95%CI 0.83 to 0.98) compared to ours with 83,247 individuals which showed a 15% in the prevention of stroke (RR 0.85, 95% CI: 0.76; 0.96, p < 0.01), all studies agreeing that the use of folic acid supplementation reduces the risk of stroke compared to placebo [7, 67]. In addition, in our subgroup analyses of participants who received a placebo, the ones receiving a regular placebo showed a significant effect in reducing stroke (RR: 0.81, 95% CI: 0.71 to 0.93) over the groups that received low-dose folic acid as placebo, this could be caused because the effect of low-dose folic acid can shadow the effect. Additionally, it indicated a significant effect, while those with only females did not. This discrepancy can be attributed to the smaller sample sizes in female-only studies, which may lead to insufficient statistical power to detect significant differences. Therefore, including both sexes in research provides a more comprehensive understanding of the effects and potential benefits of folic acid supplementation.

On the other hand, Li et al. (2016) suggested that individuals with lower baseline folate levels (< 16 nmol/L) have higher risk reduction than those with higher baseline folate level (>16 nmol/L) [7]. This is supported by Zhang N. (2024) who found that folic acid combined with vitamin B12 and B6 reduced the risk of stroke in areas without and with partial folic acid fortification [67]. We found a significant effect in patients with lower baseline folate levels as in the previously described analysis but the difference with the higher baseline folate level was not significant. Although baseline folate levels were considered, we did not propose specific clinical target levels due to insufficient specificity and consistency in the available data. Nevertheless, the potential advantages of improving folate status and/or lowering homocysteine in primary stroke prevention are further supported by the reported improvement in stroke mortality in North America and the United Kingdom associated with the introduction of required folic acid fortification [42].

In relation to the results of cardiovascular disease, both studies acknowledge that there is a small significant protective effect of folic acid supplementation in CVD, with Li et al. (2016) (RR = 0.96, 95% CI: 0.92 to 0.99, P = 0.02) and our meta-analysis (RR = 0.95, 95% CI: 0.90 to 0.99, p = 0.04) with the effect size varying between 4 and 5% [7]. As well, both studies recognize that baseline factors, such as higher homocysteine and lower folate levels, influence the effectiveness of folic acid supplementation. Our subgroup analyses showed statistically significant results with no or low heterogeneity in groups that received folic acid alone and Hcy >4 micromol/L. Furthermore, subgroup analyses by Li et al. (2016), showed significant 14% reduction in risk (RR = 0.86, 95% CI:0.79 to 0.94, P < 0.0001) in individuals without a preexisting CVD [7]. And we were capable to reduce heterogeneity to 0% excluding Armitage (2010) and Ebbing (2008) [45, 47]. The studies may have contributed to increased heterogeneity because they are influential, one is due to the effect size and the other due to its variability respectively.

Correspondingly, our meta-analysis assessed mortality by analyzing 25 studies that included 80,505 individuals, and it did not show statistical significance in reducing risk (RR = 0.98, 95% CI: 0.93 to 1.02, p = 0.25). This outcome was not evaluated by Li et al. (2006). As well, Liem et al. (2004) and Lamas G (2013) showed no beneficial effect on mortality in population of post-acute myocardial infarction who received folic acid supplementation [37, 63]. Also, Bostom et al. (2011) indicated that multivitamin with high-dose folic acid supplementation significantly reduces homocysteine levels, but it did not reduce mortality in kidney transplant recipients [51]. Similarly, Ebbing et al. (2008) found no significant benefit in reducing mortality or cardiovascular outcomes despite lowering homocysteine levels [46].

Despite of the beneficial outcomes, our meta-analyses did not show statistically significant effect in CHD, PAD, HDL and LDL levels. Similarly, studies by Vermeulen et al. (2000) and Zoungas et al. (2006), reported no significant difference in PAD levels after folic acid supplementation [21, 26]. Likewise, Liem et al. (2004), showed no significant decrease in cholesterol levels between individuals treated with folic acid in combination with statin therapy compared to those treated with statins alone [38]. These findings suggest that the protective effects of folic acid may be specific to particular cardiovascular outcomes. Notably, our study is the first to present pooled data that includes both PAD and HDL, offering valuable insights into the effects of folic acid supplementation.

Our systematic review and meta-analysis also confirmed the efficacy of folic acid supplementation in reducing homocysteine levels, similar to Tighe’s study (2011) that revealed that folic acid supplementation at doses as low as 0.2 mg/day for six months effectively lowers homocysteine concentrations [68]. However, the translation of this effect into a consistent reduction in cardiovascular events remains uncertain, except when the patient had a low baseline level of folic acid. This correlates with the findings of Yuan et al. (2021), where lower levels of homocysteine achieved by supplementing with vitamin B reduced the risk of stroke, specifically subarachnoid hemorrhage and ischemic stroke [69]. This suggests that although homocysteine is an independent risk factor for cardiovascular disease and related events, and folic acid is effective in lowering the levels, other mechanisms may be involved in the physiopathology of these conditions, which could explain the variability in findings across different studies. This is further supported by the biological role of homocysteine in cardiovascular disease, as elevated levels are known to impair endothelial function, promote oxidative stress, and increase vascular inflammation and thrombogenicity. These are factors that contribute to the development of atherosclerosis and ischemic events. Folic acid helps reduce the concentration of homocysteine by promoting its remethylation to methionine. Additionally, folic acid has been associated with improvement of endothelial function by enhancing nitric oxide availability and reducing oxidative damage. These mechanisms may help explain the protective effect observed in stroke and some cardiovascular outcomes, particularly in individuals with high homocysteine or low folate levels. In the case of stroke, especially ischemic and small-vessel strokes, the cerebral vasculature may be more sensitive to homocysteine endothelial damage. In contrast, myocardial infarction is more influenced by atherosclerotic plaques, where folic acid has less effect. This difference may explain why folic acid shows stronger preventive effects in stroke [9, 32].

The 2024 Stroke Guideline by the American Heart Association and the 2019 ACC/AHA Cardiovascular Disease Primary Prevention Guideline, consider as primary intervention the lifestyle changes, reduction of blood pressure, and cholesterol management, while considering folic acid supplementation as a secondary intervention in stroke and CVD prevention respectively, especially in individuals with elevated homocysteine levels and low folate levels [70, 71]. This being said, our study is able to provide stronger recommendations and a level of evidence for the use of folic acid in the prevention of stroke and cardiovascular disease. Additionally, our study recommends its use in the prevention of stroke and CVD, especially in patients who have elevated homocysteine levels, low folate levels, or those who don´t have a preexisting CVD disease.

There are significant implications for clinical practice considering these findings, since they may influence treatment decisions and patient management in patients with high cardiovascular risk. The results of this meta-analysis suggest that folic acid supplementation may confer selective cardiovascular benefits, particularly in reducing the risk of stroke, without significantly impacting mortality, coronary heart disease, peripheral arterial disease, or cholesterol levels. Clinicians should consider these findings when advising patients, especially those with low baseline folate levels or at high risk of stroke. Personalizing folic acid supplementation strategies is imperative, tailoring them to individual patient profiles and existing dietary folate intake. Furthermore, it is crucial for practitioners to stay informed about ongoing research and evolving guidelines that may further elucidate the role of folic acid in cardiovascular health and disease prevention.

Limitations

Our analysis, while having some limitations, has produced significant findings. Notably, over 75% of the 45 studies demonstrated a low risk of bias in critical areas such as the randomization process, deviations from intended interventions, and outcome measurement. This positive finding compensates for the high risk of bias observed in the remaining studies, particularly in the domains of missing outcome data, measurement of outcomes, and the selection of reported results. Overall, while most studies had a low risk of bias, certain areas, such as outcome measurement and selective reporting, require cautious interpretation. Our research may not have included all relevant studies worldwide due to our language restrictions, which limited our scope to English and Spanish publications. This restriction may have excluded valuable data from studies published in other languages. Given these limitations, we suggest further investigations in countries with high poverty levels, such as those in Africa and Latin America, where vitamin deficiencies are more prevalent. Additionally, funnel plots showed some asymmetry suggesting possible publication bias, but Egger’s tests were not statistically significant. Publication bias cannot be fully excluded, especially for outcomes with few studies where formal assessment wasn’t possible such as PAD; HDL and LDL levels. Another limitation is the lack of consensus among authors in defining the outcome measures, which may have introduced variability in the interpretation of results across studies. Additionally, the concomitant use of folic acid with other medications could have confounded the effect size attributed solely to folic acid. Dietary intake of folic acid was not standardized across participants, potentially influencing baseline levels and modifying treatment effects. These factors may contribute to heterogeneity and should be considered when interpreting our findings. Future trials should control for baseline folate status and assess folic acid as a monotherapy to better isolate its effects. Additionally, expanding the study area to include diverse regions would provide a broader understanding and potentially lead to more comprehensive and robust conclusions.

Conclusion

In conclusion, this systematic review and meta-analysis has highlighted the selective benefits of folic acid supplementation cardiovascular disease notably in reducing stroke risk and, to a lesser extent, overall cardiovascular disease. This is especially considered in patients who have elevated homocysteine levels (> 4 micromol/L), low folate levels (< 16 nmol/L) or those who do not have a preexisting CVD disease. In contrast, its effects on mortality, coronary heart disease, peripheral arterial disease, LDL and HDL remain inconclusive. Given the variability and specific contexts in which folic acid demonstrates benefits, future research should aim to delineate its role in cardiovascular health further, considering individual patient profiles and demographic characteristics particularly in underrepresented subgroups, such as older adults, individuals with renal impairment, and populations without folate food fortification. This could potentially enhance personalized medical guidelines and preventive strategies for stroke tailored to diverse populations’ nutritional and biochemical needs.

Abbreviations

ABI

Ankle-Brachial Index

BMI

Body Mass Index

BP

Blood Pressure

CHO

Carbohydrate

CI

Confidence Interval

CINAHL

Cumulative Index to Nursing and Allied Health Literature

CV

Coefficient of Variation

CVD

Cardiovascular Disease

DBP

Diastolic Blood Pressure

EMBASE

Excerpta Medica Database

ES

Effect Size

G

Grams

HDL

High-Density Lipoprotein

I²

I-squared statistic

IQR

Interquartile Range

LL

Lower Limb

MD

Mean Difference

mmHg

Millimeters of Mercury

NR

Not Reported

PAD

Peripheral Arterial Disease

PRISMA

Preferred Reporting Items for Systematic Reviews and Meta-Analyses

PROSPERO

International Prospective Register of Systematic Reviews

RE

Random Effects

RI

Resistance Index

RR

Relative Risk

SBP

Systolic Blood Pressure

SD

Standard Deviation

SMD

Standardized Mean Difference

TC

Total Cholesterol

TG

Triglycerides

VO₂max

Maximal Oxygen Uptake

WMD

Weighted Mean Difference

Authors’ contributions

“Conceptualization, E.C.; Methodology, E.C; Software, E.C, J,A, A.A.; Validation, E.C, PG.; Formal analysis, E.C, P,G.; Investigation, P.G, E.C, M.G, J.A, J.M, B.S and K.C; Resources, E.C, P.G, J.A, and A.A.; Data curation: E.C, A.A.; Writing—original draft preparation, P.G, J.A, J.C, A.A, M.G, J.M, B.S, and K.C.; Writing—review and editing, P.G, E.C, J.A, J.C, A.A, B.S, K.C, G.F, and C.S; Visualization, P.G.; Supervision, E.C.‘’.

Funding

“This research received no external funding”.

Data availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.