Palpitations, defined as abnormally rapid or irregular heartbeats, may present as skipped beats, fluttering, or a pounding sensation in the chest or neck. Although often benign, they can indicate life-threatening conditions. Palpitations can occur due to structural heart disease or systemic metabolic disorders; however, current knowledge regarding their effects on the autonomic nervous system is limited [1].

Heart rate variability (HRV) is a noninvasive marker of autonomic nervous system function. Reduced HRV is associated with adverse cardiovascular outcomes, including arrhythmias and increased mortality risk [2]. Sex differences in HRV exist, with women showing higher parasympathetic indices and men showing greater sympathetic modulation, indicating sex-specific autonomic regulation [3]. Recent studies have also highlighted autonomic dysfunction in outpatient populations with systemic or post-infectious conditions, emphasizing the clinical relevance of HRV assessment beyond overt cardiovascular disease [4]. Beyond autonomic tone, micronutrient status may also influence HRV. Vitamin B12 and folate are essential cofactors in one-carbon metabolism, and deficiencies can impair hematological and neurological function, potentially altering cardiac autonomic regulation [5]. In functional iron deficiency (FID), inflammation-induced hepcidin upregulation restricts iron availability for erythropoiesis despite adequate or increased iron stores. Unlike absolute iron deficiency, iron-binding capacity (IBC) does not exhibit a compensatory increase and should be interpreted in conjunction with other iron indices and the inflammatory milieu [6]. Combined deficiencies of Vitamin B12 and iron are frequently observed in clinical populations and are associated with distinct hematological and metabolic alterations that may have downstream effects on autonomic regulation [7]. Similarly, it has been shown that HRV is reduced in iron-deficient patients, and parasympathetic nervous system effects are impaired in iron-deficient individuals [8]. The common finding across these studies is that nutritional deficiencies adversely affect autonomic nervous system function and reduce HRV [9, 10, 11, 12]. Recent evidence highlights that sex-specific differences in vitamin metabolism and oxidative stress regulation may contribute to cardiometabolic health disparities, underscoring the importance of integrating nutritional biomarkers into cardiovascular risk assessment [13].

Despite these insights, few studies have simultaneously examined HRV in relation to sex differences and micronutrient status. Understanding these associations may provide novel perspectives on autonomic regulation and its interaction with nutritional deficiencies, particularly in patients with palpitations. Among HRV indices, standard deviation of all normal-to-normal intervals (SDNN) was selected as the primary outcome because it reflects overall autonomic modulation by integrating both sympathetic and parasympathetic influences and is widely accepted as a robust global measure of HRV in clinical and epidemiological studies. This study aimed to investigate sex-specific alterations in HRV and their associations with serum Vitamin B12, folate, and iron levels, thereby contributing to a more comprehensive understanding of cardiovascular risk stratification.

In the present study, we observed marked sex-specific differences in HRV parameters, with women demonstrating significantly lower time- and frequency-domain indices (SDNN, rMSSD, pNN50, Low-Frequency (LF) Power, and Very-Low-Frequency (VLF) Power) than men. This is consistent with prior observations of sex differences in autonomic cardiac regulation, in which men typically exhibit higher overall HRV, reflecting greater autonomic flexibility [3, 14]. Multivariable regression analysis further confirmed that male sex was independently associated with higher SDNN ($\beta$ = 0.467, p 0.001, Fig. 1), whereas SDNN decreased with advancing age ($\beta$ = –0.269, p 0.001). Umetani et al. [15] reported a progressive age-related decline in multiple time-domain HRV measures, including SDNN, across nine decades in healthy subjects, underscoring the physiological reduction in autonomic modulation with aging.

Iron deficiency was significantly more prevalent in women (23% vs. 1.9%, p 0.001), accompanied by lower ferritin, serum iron, and hemoglobin levels and higher IBC. Importantly, IBC may capture early or subclinical disturbances in iron availability that are not fully reflected by ferritin levels alone, thereby providing additional insight into iron-related autonomic dysregulation even in the absence of overt anemia. Spearman’s correlation revealed positive associations between HRV parameters and iron markers (ferritin and serum iron), which were notably stronger in women. These findings align with evidence linking iron deficiency anemia to impaired autonomic function, primarily through reduced parasympathetic activity and sympathetic predominance [8, 16]. For example, Tuncer et al. [16] demonstrated lower HRV in patients with iron deficiency anemia than in controls, attributing this to hypoxia-induced sympathetic activation. Similarly, in patients with coronary heart disease or chronic anemia (e.g., thalassemia), anemia is associated with diminished HRV, potentially mediating a heightened cardiovascular risk [17, 18]. Higher IBC emerged as an independent negative predictor of SDNN ($\beta$ = –0.377, p 0.001), further supporting the role of latent iron deficiency in the autonomic dysregulation. Although some correlations were statistically significant, the magnitude of the other relationships was modest, and some of the findings should be interpreted as weak clinical correlations rather than strong relationships. The stronger correlations observed in women may partly reflect menstrual blood loss and hormonal influences on iron homeostasis, exacerbating their vulnerability to autonomic dysfunction. Elevated IBC may reflect reduced bioavailable iron, potentially impairing oxygen transport, mitochondrial oxidative phosphorylation, and cellular energy production. These alterations may activate compensatory neurohumoral pathways and sympathetic drive, thereby contributing to autonomic imbalance and reduced HRV [19].

Likewise, folate deficiency was more common in women (5.6% vs. 1.4%, p = 0.034), with positive correlations between HRV indices and folate levels, which were stronger in females than in males. Folate independently predicted higher SDNN in the regression analysis ($\beta$ = 0.117, p = 0.037). Vitamin B12 deficiency was associated with weaker but positive correlations with HRV. These nutrient-HRV links are supported by studies indicating that Vitamin B12 and folate deficiencies disrupt autonomic balance, possibly via hyperhomocysteinemia, oxidative stress, or impaired neuronal myelination [11, 12]. Sucharita et al. [20] found reduced HRV in Vitamin B12-deficient patients, reversible with replacement therapy. Supplementation trials have similarly shown improvements in HRV parameters following B12 repletion in elderly participants. Folate deficiency is associated with impaired nitric oxide bioavailability, endothelial dysfunction, and increased sympathetic drive. Although direct evidence for the effect of folate supplementation on HRV is limited, its role in homocysteine metabolism suggests that it shares pathways with B12 in maintaining the vagal tone [21, 22].

Mechanistically, these micronutrients contribute to erythropoiesis, redox balance, and neurotransmitter synthesis, and deficiencies may promote sympathetic dominance, as evidenced by higher LF/HF ratios in deficient states. Sex disparities likely stem from reproductive factors that increase the deficiency risk in women, amplifying the autonomic effects [19]. Despite the non-significant association with CRP, subclinical inflammation cannot be fully excluded.

This study has several limitations related to its design, patient population, and single-center nature.

● Its retrospective design precludes causal inference, and unmeasured confounders may still be present despite the strict exclusion criteria.

● Although ferritin, iron, and inflammatory markers were evaluated, ferritin can be influenced by subclinical inflammation, and more sensitive inflammatory biomarkers (e.g., IL-6 and hepcidin) were not available in the dataset used.

● In addition, detailed information on dietary intake and micronutrient supplementation was not available, which may have influenced circulating iron, folate, and Vitamin B12 levels.

● HRV parameters were derived from 24-hour Holter recordings, which provide a robust assessment of autonomic function but may still be influenced by daily activity patterns, sleep quality, and psychological stress, factors that cannot be fully standardized or quantified retrospectively.

● The study population consisted of individuals presenting with palpitations, which may limit the generalizability of the results to asymptomatic or community-based populations. The study population may not be representative of the general population, limiting the generalizability of the findings.

● Finally, although the sample size was adequate for multivariable modeling, the inclusion of additional biochemical markers or longitudinal follow-up could further strengthen the mechanistic interpretation of the findings.

In this cohort of adults presenting with palpitations, SDNN, a key marker of global autonomic modulation, was strongly influenced by age, sex, and biochemical indices of iron metabolism. Male sex and younger age were associated with higher SDNN values, underscoring the importance of demographic stratification when interpreting HRV metrics in clinical settings. Although ferritin, serum iron, folate, and Vitamin B12 showed significant correlations with HRV parameters, only IBC and folate remained independent predictors in the multivariate analysis. These findings suggest that alterations in iron metabolism, reflected by elevated IBC, may impair autonomic regulation even in the absence of overt anemia, whereas adequate folate status may support healthier autonomic function.

Overall, our results highlight the need to consider demographic, biochemical, and nutritional factors when evaluating HRV in patients with palpitations. Identifying modifiable biochemical factors that influence HRV may offer opportunities for early intervention in patients with palpitations. Moreover, the strong effects of age and sex emphasize the need for demographic adjustments when interpreting HRV metrics in clinical settings. Given the retrospective observational design, the associations observed in this study do not imply causality. Therefore, no conclusions can be drawn regarding the potential benefits of iron or folate supplementation, which should be evaluated in future prospective and interventional studies. Future prospective studies are warranted to clarify the mechanistic pathways linking iron homeostasis and autonomic balance and to determine whether correcting iron handling-abnormalities or folate deficiency can improve HRV and clinical outcomes.

ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; BUN, Blood Urea Nitrogen; CRE, Creatinine; CRP, C-Reactive Protein; ECG, Electrocardiography; GLU, Glucose; HB, Hemoglobin; HF, High-Frequency; HR, Heart Rate; HRV, Heart Rate Variability; IBC, Iron-Binding Capacity; LF, Low-Frequency; LF/HF, Low-to-High Frequency; NN, Normal-to-Normal Intervals; Mean_HR, Mean Heart Rate; pNN50, Percentage of successive NN Intervals differing by more than 50 ms; rMSSD, root Mean Square of Successive Differences; SDNN, Standard Deviation of all NN Intervals; SDNN-index, Mean of the Standard Deviations of NN intervals for all 5-minute segments; TSH, Thyroid-Stimulating Hormone; VLF, Very-Low-Frequency; WBC, White Blood Cell Count.

The data used in this study are available upon reasonable requests.

The manuscript was conceived by MÖ, SÖ. The data were collected and the images and tables were prepared by MÖ and SÖ. MÖ, SÖ, TM, and Mİ were responsible for data analysis, statistical analysis, and interpretation of the results. The manuscript was written by MÖ, SÖ, TM, and Mİ. TM and Mİ critically revised the manuscript for important intellectual content. The entire process was supervised by MÖ and SÖ. The final version of the manuscript was carefully reviewed and approved by all authors, and all authors agree to be accountable for all aspects of the work.

The study was conducted in accordance with the principles of the Helsinki Declaration and approved by the Konya City Hospital Ethics Committee (decision number 225/2025, dated November 10, 2025, meeting number 2025/11). Due to the retrospective design of the study, obtaining informed consent was not deemed necessary.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.

The graphical abstract was prepared using GPT-5.2 and OpenAI. The authors performed the study design, data analysis, and interpretation of results.