Open Veterinary Journal, (2026), Vol. 16(4): 2193-2202

Research Article

10.5455/OVJ.2026.v16.i4.21

Heart rate variability in horses with naturally occurring gastric ulcers: Response to omeprazole and sucralfate treatment

Dania Cingottini, Francesca Bindi, Giulia Sala^*, Benedetta Sorvillo, Alessia Galiano,
Francesca Bonelli and Micaela Sgorbini

Department of Veterinary Sciences, Veterinary Teaching Hospital, University of Pisa, Pisa, Italy

*Corresponding Author: Giulia Sala. Department of Veterinary Sciences, Veterinary Teaching Hospital, University of Pisa, Pisa, Italy. Email: giulia.sala [at] unipi.it

Submitted: 18/11/2025 Revised: 11/03/2026 Accepted: 24/03/2026 Published: 30/04/2026

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Abstract

Background: Equine gastric ulcer syndrome (EGUS) is the most common disease affecting the equine stomach. Heart rate variability (HRV) describes and quantifies the variability between consecutive heartbeats due to the neurohormonal regulation of the heart. It has been considered a non-invasive method to detect autonomic tone changes that can be influenced by pain and disease.

Aim: This study aimed to assess the HRV indices in horses affected by EGUS submitted or not to treatment.

Methods: A cohort of 45 mares underwent baseline gastroscopy (T0); 24/45 mares diagnosed with Equine Squamous Gastric Disease (ESGD) graded 2 out of 4 or greater were included and assigned to treated (T, n=12) or non-treated (NT, n=12) groups. The T group received omeprazole and sucralfate for 28 days. Heart rate (HR) was recorded using an HR monitor before the treatment (T0) and at the end of the treatment (T29). R-R intervals were analyzed using commercial software to obtain time-domain, frequency-domain, and geometric indices for the HRV parameters. Gastroscopies were performed at the end of the treatment (T29) for both groups and at T14 for the NT group.

Results: At T0, ESGD at least 2/4 score was diagnosed in all the horses included, while Equine Glandular Gastric Disease (EGGD) at least 1/4 score was diagnosed in 8/12 and 12/12 horses in the NT and T groups, respectively. No significant differences were observed in HRV indices within or between groups over time. The treatment significantly reduced ESGD scores (p=0.018), but not the EGGD scores (p=0.573) in the T group.

Conclusion: Although HRV did not change significantly post-treatment, it appears to be a promising tool for assessing autonomic function in EGUS.

Keywords: Equine, Equine glandular gastric disease, Equine squamous gastric disease, Heart rate variability, Ulcers.

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Introduction

Equine gastric ulcer syndrome (EGUS) is the most common disease affecting the horse's stomach (Sykes et al., 2015). Although EGUS is particularly common in sport horses (37% up to 80%–100% in training) (Sykes et al., 2015; Vokes et al., 2023), a high prevalence has also been reported in pleasure horses (40%) (Niedźwiedź et al., 2013; Busechian et al., 2024), breeding mares (71%) (le Jeune et al., 2009; Busechian et al., 2024), and young horses (up to 57%) (Hewetson et al., 2018; van den Boom and Kranenburg, 2024).

The term EGUS encompasses both Equine Squamous Gastric Disease (ESGD) and Equine Glandular Gastric Disease (EGGD), depending on the distribution of the lesions. ESGD is primarily caused by mucosal exposure to gastric and volatile fatty acids (Sykes et al., 2015; Lorenzo-Figueras and Merritt, 2002; van den Boom, 2022). On the other hand, the pathophysiology of glandular ulceration is unclear, and the breakdown of the natural defense mechanisms of the mucosa is believed to be involved (Sykes et al., 2015). In both cases, stress and feeding management have been reported to be predisposing factors for gastric ulceration (Luthersson et al., 2009; Sykes et al., 2015, 2019).

Clinical signs are usually non-specific and include poor performance, abdominal pain, weight loss, inappetence, and behavioral changes (Dukti et al., 2006; Nieto et al., 2009; Sykes et al., 2015; Lo Feudo et al., 2022; Vokes et al., 2023). In some cases, there are no clinical signs even though gastric ulcers are present and visible on gastroscopy (Sykes et al., 2015, 2019; Spanton, 2024).

The treatment of ESGD focuses primarily on reducing gastric acidity (Sykes et al., 2015). Proton pump inhibitors (PPIs) and H2-receptor antagonists are the most used classes of drugs (Sykes et al., 2015). Among PPIs, omeprazole has been the most extensively studied and is available in various formulations suitable for equine use, including gastro-resistant granules, buffered paste, and intramuscular preparations (Sykes et al., 2014, 2015, 2017a; Gough et al., 2020; Busechian et al., 2023). Regarding H2-receptor antagonists, ranitidine is efficacious but generally considered a second-line treatment option (Sykes et al., 2015; Vokes et al., 2023).

In contrast, the pathophysiology of EGGD is poorly understood, thus making treatment more challenging (Sykes et al., 2015; Rendle et al., 2018). A combination of omeprazole and sucralfate is currently recommended to promote mucosal healing, with treatment typically continued for up to 8 weeks before considering any modifications (Sykes et al., 2015; Rendle et al., 2018; Banse and Andrews, 2019). Additionally, misoprostol—a prostaglandin analogue—is effective in healing glandular lesions and can be used in refractory cases (Varley et al., 2019).

Heart rate variability (HRV) is a non-invasive means to assess the sympathovagal balance of the autonomic nervous system (ANS) by quantification of the heart rate (HR) beat-to-beat variations (Bowen, 2010; Vitale et al., 2013; Stucke et al., 2015). HRV is influenced by factors such as stress, pain, and diseases (Rietmann et al., 2004a; von Borrell et al., 2007; Stucke et al., 2015; Perron et al., 2023). A decrease in variability is correlated with reduced parasympathetic tone and increased sympathetic tone. While this autonomic pattern may occur during physiological conditions such as exercise or acute stress, it may also be indicative of underlying disease processes, including gastric ulceration (Mitchell and Schwarzwald, 2021).

Gastrointestinal function and secretion are closely regulated by the autonomic nervous system, and alterations in sympathovagal balance may influence gastric physiology (Stucke et al., 2015; Louie et al., 2023). Moreover, the pain associated with gastric ulcers has the potential to further modulate autonomic tone, leading to measurable changes in HRV (Louie et al., 2023). In human medicine, peptic ulcer disease has been associated with increased sympathetic activity, as assessed through HRV analysis, supporting the link between gastric pathology, pain, and autonomic dysfunction (Nada et al., 2001). In equine medicine, recent experimental studies have demonstrated a correlation between HRV indices and the presence of gastric ulcers (Louie et al., 2023; Perron et al., 2023). In these studies, ulcers were experimentally induced, and changes in HRV were observed, suggesting that HRV may serve as a proxy measure of gastric pain and autonomic imbalance. Particularly, a decrease in the sympathetic tone and a concomitant increase in parasympathetic tone were observed in horses treated with omeprazole, compared to untreated horses (Louie et al., 2023). Although gastroscopy remains the gold standard for the diagnosis of EGUS (Sykes et al., 2015), its cost and the perception of invasiveness often limit its use in clinical practice. In this context, HRV analysis may represent a useful, non-invasive tool for screening horses that would benefit from further diagnostic investigation via gastroscopy, but also by monitoring autonomic changes over time and potentially assessing treatment efficacy.

The aim of this study was to evaluate HRV indices in horses naturally affected by EGUS and subsequently assigned to either treatment or no-treatment groups.

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Materials and Methods

Animals and experimental design

The present study is a prospective clinical study. The Veterinary Teaching Hospital (VTH) at the University of Pisa owns a cohort of 80 mares that were used as recipients in a February-August commercial embryo transfer program. The mares are housed in groups of 10–15 animals in 25 × 25 m sand paddocks. Mares are provided with ad libitum access to hay and water and are supplemented with grain once daily at 8:00 a.m. at a dose of 1 g/kg/day (Equi-fioc, Molitoria Val di Serchio, Lucca, Italy).

From September to January, non-pregnant mares typically undergo clinical examinations and general health checks. Between November 2024 and January 2025, a cohort of 45 non-pregnant mares housed at the VTH underwent gastrointestinal health assessments, including gastroscopy (T0).

The procedure revealed that 24/45 mares were affected by ESGD, scored at least 2/4, and/or EGGD, defined as any mucosal alteration ranging from hyperemia to ulceration.

The cohort of 24 mares affected by EGGD and/or EGGD was assigned to treatment (T; n=12) and non-treatment groups (NT; n=12) in a non-randomized manner based on the severity of lesions. Horses with severe lesions [EGGD score 4/4 or total Equine Gastric Disease (EGD) score >6/8] were automatically assigned to the T group.

The T group received gastro-resistant omeprazole granules (Gaster®, ACME S.r.l., Italy) at a dose of 4 mg/kg once daily, and sucralfate (Sucralfato DOC Generici, DOC Generici S.r.l, Italy) at 12 mg/kg every 12 hours for 28 consecutive days. At 6:00 a.m. (2 hours before grain feeding), both omeprazole and sucralfate were administered orally and mixed with a small amount of mash (Sykes et al., 2015; Rendle et al., 2018; Vokes et al., 2023). Then, at 6:00 p.m., sucralfate was administered orally, and mixed with mash. Gastroscopy was repeated at the end of the treatment (T29). Additionally, in the NT group, mares underwent a gastroscopy at T14 and were excluded if lesions had worsened.

HRV assessment was performed at T0 (before starting the treatment) and T28 (at the end of the treatment). HRV recordings at T28 were carried out in the morning, before the beginning of the fasting period required for gastroscopy, which was performed the following day.

Gastroscopic examination and scoring

Gastroscopy was performed using a 300–-cm-long endoscope (60130PKS, Karl Storz Endoscope, Tuttlingen, Germany) and a portable processor (Gastropack, Karl Storz, Germany, and Telepack Vet, Karl Storz, Germany). Horses were fasted for 16 hours, and water was withheld for 6 hours prior to the procedure. The horses were restrained in a stock, sedated with detomidine (Dorum®, ACME S.r.l., Italy) at a dosage of 0.012 mg/kg, IV, and butorphanol (Nargesic®, ACME S.r.l., Italy) at a dosage of 0.025 mg/kg, IV. A nose twitch was also applied. The gastroscope was passed through the ventral nasal meatus into the oesophagus and advanced into the stomach. The stomach was insufflated with air until the internal stomach folds appeared flattened. Feed material on the mucosa was washed away using water through the biopsy channel of the endoscope to visualize the entire squamous and glandular part of the stomach. Videos were recorded for subsequent evaluation of lesions and data storage.

The presence and severity of lesions on squamous and glandular mucosa were assessed according to the ECEIM consensus statement guidelines (Sykes et al., 2015). Additionally, lesions on glandular mucosa were scored using a 0–4 grading system (0=no lesions, 4=deep or fibrinosuppurative ulcerations) (Rendle et al., 2018; Wise et al., 2021; Pineau et al., 2024). A total EGD score was calculated by summing the squamous and glandular scores (Pineau et al., 2024).

Heart rate variability assessment and analysis

The recordings for HR variability assessment were performed for at least one hour with the horse standing alone, without any restraint, in a 4 × 4 m stall, located in a building in the university hospital. Each mare underwent an 18–20-hour acclimatization period, without data collection, to reduce the risk of stress influence on the HRV indices (Vitale et al., 2013). HR was recorded in the morning, from 8:00 to 12:00, at T0 (before starting the treatment) and T28 (at the end of the treatment).

Heart rate was recorded using an HR monitor (Polar Equine H10 HR Sensor Trotters, Polar Electro Oy, Kempele, Finland) attached to a chest belt with electrodes (Polar EquineBelt, Polar Electro Oy, Kempele, Finland), as previously reported (Visser et al., 2002; Schmidt et al., 2010; Stucke et al., 2015; Ille et al., 2014; McDuffee et al., 2019; Mitchell and Schwarzwald, 2021; Vitale et al., 2021, 2024). The HR monitor was connected to a sports watch (Polar Vintage M2, Polar Electro Oy, Kempele, Finland), secured to the chest belt or the halter. After the recordings, data were extracted as R-R intervals from the watch using specific Polar FlowSync v 4 (Polar Electro Oy, Kempele, Finland). After applying the artifact removal option (medium setting), the central 5-minute period of the 1-hour recording was selected for HRV analysis, using Kubios HRV (Kubios HRV Scientific 4.1.0, Kubios Oy, Kuopio, Finland) (Malik et al., 1996; Bowen, 2010; Vitale et al., 2013; Stucke et al., 2015; McConachie et al., 2016; Vitale et al., 2020).

In the time domain analysis, mean R-R (mR-R, ms), standard deviation of R-R intervals (SDRR, ms), and root mean square of successive squared RR differences (RMSSD, ms) were assessed. In the frequency-domain analysis, the low-frequency (LF) and high-frequency (HF) peaks (nu), LF and HF power (nu), and LF/HF ratio were assessed. LF and HF bands were set at 0.04–0.15 Hz and 0.15–0.4 Hz, respectively. Geometric indices SD1 (standard deviation of the instantaneous beat-to-beat variability data) and SD2 (standard deviation of the continuous long-term variability) were also assessed.

Statistical analysis

Regarding statistical analysis, an a priori power analysis was conducted using G*Power v. 3.1 to estimate the required sample size for detecting an interaction effect in a mixed-design analysis of variance (ANOVA). The analysis was based on an effect size of 0.5, a significance level (α) of 0.05, and a statistical power (1–β) of 0.95. Although pilot studies yielded an effect size of approximately 1.4 (Perron et al., 2023), the current study involves a spontaneously occurring pathology rather than experimentally induced conditions. A less pronounced severity and greater variability are therefore expected. To adopt a more conservative and realistic approach, an effect size of 0.5 was used to calculate the sample size. Based on these criteria, the total sample size required was 22 horses. The minimum number of 22 animals was increased by 10% to account for potential horses lost in follow-up. The total number of animals required for the study was thus 24.

The Shapiro–Wilk test was used to assess the normality of continuous variables. Normally distributed continuous variables were expressed as mean ± standard deviation (SD), whereas non-normally distributed data were expressed as median, 25th, and 75th percentiles. Categorical variables were expressed as frequency and percentage. All HRV parameters resulted in normal distributions, and only age was not normally distributed.

At baseline (T0), equality of the distribution of ESGD, EGGD, and EGD scores between treated and non-treated groups was assessed using Pearson’s chi-square test. Because ulcer scores (ESGD, EGGD, and EGD) are ordinal variables repeatedly measured over time, changes in scores within and between groups were analysed using generalized estimating equations (GEE) with an ordinal logit link function. The model included time and treatment group as fixed effects and horse as the subject variable, with an exchangeable correlation structure. For the HRV indices, a one-way ANOVA test was used to assess the changes within groups over time, while a generalized mixed model, with treatment as the fixed effect, was used to assess differences in HRV indices and ulcer score when comparing groups over time. SPSS 29.0 for Mac (IBM, Armonk, USA) was used for statistical analysis. Statistical significance of p-values was set at 0.05.

Ethical approval

The study was approved by the Ethical Committee of the University of Pisa, n. 7/24, 31/01/2024.

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Results

Horses in the NT group and T group had a median age of 9.5 (3–19) and 12 (3–23) years, respectively. No statistical differences were found for ESGD (p=0.822), EGGD (p=0.151), and EGD (p=0.659) scores at T0 between the two groups. One/12 horse in the NT group was excluded after T14 gastroscopy, due to worsening of the gastric ulcers, and underwent treatment. 

Table 1 reports the ESGD, EGGD, and EGD scores, expressed as frequency and percentage, divided by time and T and NT groups. According to the ordinal GEE model, both time and treatment had a significant effect on ESGD scores (p=0.022; p=0.018, respectively). Scores significantly improved over time across all horses, and treated horses showed consistently lower ESGD scores compared with non-treated ones. In particular, the odds ratio for the time effect indicated that, compared with T28, the odds of exhibiting higher ESGD scores were 2.27 (95% CI: 0.74–7.00) at T0 and 0.56 (95% CI: 0.21–1.51) at T14. Regarding treatment, non-treated horses had 3.34 times higher odds of exhibiting more severe ESGD lesions compared with treated horses (95% CI: 1.23–9.07). According to the ordinal GEE model, no significant effects of time or treatment were detected for EGGD scores (p=0.735; p=0.573, respectively). Neither time nor group influenced the severity or distribution of glandular gastric lesions; however, in the T group, 6/12 horses showed an improvement in at least one grade in the EGGD score, while 4/12 horses maintained the same score, and 2/12 horses showed a worsening of lesions by 1 grade, after 28 days of treatment.  Regarding the EGD score, the ordinal GEE model did not detect a statistically significant effect for either time (p=0.111) or treatment (p=0.438).

Table 1. Frequency (Freq) and percentage (Perc %) of categorical variables divided by time and treated (T)/non-treated (NT) group, regarding Equine Squamous Gastric Disease (ESGD) and Equine Glandular Gastric Disease (EGGD).

Table 2 reports the HRV indices, expressed as mean ± standard deviation, minimum value, and maximum value. 

Table 2. Descriptive statistics heart rate variability (HRV): continuous variables divided by time and T/NT group. SD: standard deviation; NT: not treated; T: treated.

No significant differences were found in the T group or in the NT group after treatment. Changes in time evaluated using a mixed model showed no differences in HRV indices.

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Discussion

This study evaluated the differences in HRV indices in horses naturally affected by gastric ulcers, comparing those that received treatment to those that did not.

Overall, no significant changes in HRV indices were observed after treatment in the T group compared to the NT group. In the T group, a significant improvement in ESGD was noted, while EGGD and EGD showed no significant improvement after treatment.

In previous studies (Louie et al., 2023; Perron et al., 2023), a correlation between HRV and gastric ulcers was found. An experimental design was used in both previous studies by inducing gastric ulcers in horses using specific protocols (Murray, 1992, 1994). In the study by Louie et al. (2023), which involved nine geldings, an increase in R-R intervals and a decrease in LF/HF ratio in horses treated with omeprazole were found after the induction of ulcers. These suggest a reduction in sympathetic tone because of the reduction in pain.

An increase in HR, a significant decrease in SDRR and RMSSD, and an increase in the LF/HF ratio were found by Perron et al. (2023) in horses with more severe gastric ulcers. This was based on a study of eight horses, both mares and geldings, thus suggesting an increase in sympathetic tone correlated to the increase in the severity of the lesions (Perron et al., 2023).

In this study, no significant changes in HRV indices after treatment for gastric ulcers were found. This discrepancy might be due to differences in study design and ulcer etiology. In the present protocol, the horses were naturally affected by gastric ulcers, while in both previous studies (Louie et al., 2023; Perron et al., 2023), the ulcers were experimentally induced. Unlike previous studies, which evaluated acutely induced lesions (Louie et al., 2023; Perron et al., 2023), the present investigation focused on naturally occurring gastric ulcers, which are typically heterogeneous in origin, severity, and chronicity (Bell et al., 2007; Sykes et al., 2015). This heterogeneity may influence pain perception and autonomic responses, potentially reducing detectable HRV changes. Although EGUS is generally considered a painful condition, based on associated clinical signs such as altered performance, behavioural changes, girth sensitivity, and pre-feeding aggressiveness (Vatistas et al., 1999; Millares-Ramirez and Le Jeune, 2019; Ferlini Agne et al., 2023; Pineau et al., 2024), these signs may be intermittent and context-dependent, such as during exercise or periods of low gastric pH (Sykes et al., 2015). Definitive evidence of pain associated with gastric ulcers in horses is still lacking (Sykes et al., 2015), although previous studies in human medicine have demonstrated pain associated with peptic ulcers, particularly during acid exposure (Kang et al., 1986). Another possible explanation for the absence of HRV differences between treated and untreated horses could be the presence of compensatory adaptation within the ANS. In chronic conditions, the equine ANS may maintain sympathovagal balance to mitigate ongoing stress. This adaptation may prevent overt HRV changes by enhancing parasympathetic tone or modulating sympathetic activity, preserving overall cardiac stability even as ulcers persist (von Borrell et al., 2007; Louie et al., 2023; Perron et al., 2023; Huangsaksri et al., 2024; Daibes et al., 2025).

Heart rate variability was obtained with horses restrained in stalls. Although the mares were normally housed in groups in paddocks, they were moved to stalls 18–20 hours before HRV assessment to allow adequate acclimatization (Vitale et al., 2013). This management choice aimed to reduce movement-related artifacts while avoiding excessive restraint, which may itself induce stress (Stucke et al., 2015). Despite the acclimatization period and visual contact with conspecifics, stall housing differed from the horses’ usual environment and may have influenced stress levels during recordings.

In the present study, an improvement in the ESGD score was observed in the T group after treatment with omeprazole. This is consistent with the literature and supports the efficacy of omeprazole in treating squamous ulceration (Sykes et al., 2015).

For EGGD, no statistically significant improvement was observed after treatment. This could be due to several factors. First, the pathophysiology of EGGD is still unclear (Sykes et al., 2015; Rendle et al., 2018), and the treatment efficacy is variable. Reported healing rates range from 67% (Hepburn and Proudman, 2014) to 22% (Varley et al., 2019), partly due to different definitions of “healing.” In the former study, grade 1 lesions (hyperemia of mucosa) were considered healed, whereas the latter study classified only complete resolution (grade 0) as healing. In this study, healing was defined as grade 0. Second, the lack of improvement in EGGD might be due to the administration protocol of omeprazole and sucralfate. Omeprazole seems to be more effective when administered after 8 hours of fasting, and feed should not be administered for at least 30 minutes after omeprazole administration. Indeed, PPIs are inhibited by the presence of food within the stomach (Sykes et al., 2015; Rendle et al., 2018). Ad libitum access to forage appears to reduce the bioavailability of omeprazole and its capacity to raise gastric pH (Merritt et al., 2003; Sykes et al., 2017b). Additionally, a 30-minute interval between omeprazole and sucralfate administration is recommended to decrease the risk of impaired bioavailability (Sykes et al., 2015; Rendle et al., 2018; Banse and Andrews, 2019). Third, treatment duration was limited to 28 days, which represents the minimum recommended period of treatment before reassessing ulcer severity. A longer treatment duration, up to 6–8 weeks, may have resulted in greater improvement or complete healing of the lesions (Sykes et al., 2015; Rendle et al., 2018).

The lack of EGGD improvement after 28 days may also explain the absence of detectable HRV changes following treatment. The treatment duration period adopted in this study was based on current consensus recommendations indicating a minimum treatment period of 28 days (Sykes et al., 2015) and on previous HRV-related studies (Louie et al., 2023). Therefore, the present investigation should be considered an initial clinical evaluation rather than a definitive assessment of HRV response to EGUS treatment.

This clinical research has several limitations. First, omeprazole and sucralfate were administered simultaneously due to herd management constraints. The mares were housed in groups in paddocks, and it was not feasible to administer treatments to 12 horses across different paddocks while maintaining the recommended 30-minute interval in the morning. Additionally, horses had ad libitum access to hay, and fasting before and after omeprazole administration was not guaranteed. However, most horses tend to rest overnight (from approximately 10:00 p.m. until sunrise) and consume minimal forage during this period, even when it is freely available (Ellis et al., 2015; Rendle et al., 2018; Vokes et al., 2023). In the present study, omeprazole was administered at 6:00 a.m.,2 hours before grain feeding (at 8:00 a.m.), when horses likely had a partially empty stomach. This suboptimal administration protocol represents a limitation of the study. Second, the treatment duration was limited to 4 weeks. Healing rates, particularly for EGGD, are reported to improve when treatment is extended for up to 8 weeks (Sykes et al., 2015; Rendle et al., 2018). If ulcers remain unhealed after this period, a change in therapeutic approach is recommended, including switching to misoprostol as a monotherapy. In the present study, omeprazole and sucralfate were administered for only 4 weeks; therefore, extending the treatment period or modifying the therapeutic protocol, transitioning to misoprostol may have resulted in complete healing of the EGGD lesions (Varley et al., 2019), potentially leading to detectable changes in HRV parameters. Third, the assignment of horses to the T and NT groups was not randomized, but rather based on the severity of lesions, which potentially introduced a selection bias. This non-randomized assignment to T or NT groups was a welfare-driven decision, in accordance with the recommendations of the Ethical Committee of the University of Pisa. However, no statistical differences were found between the 2 groups at T0 for ESGD, EGGD, and EGD scores, which partially mitigates this concern.

Furthermore, the study design did not include a healthy control group, as only horses affected by gastric ulcers were enrolled. The inclusion of a healthy control group may have allowed clearer differentiation of HRV patterns between EGUS-affected and unaffected horses, regardless of treatment.

In addition, horses in the NT group did not receive a placebo, and only mares were included in the study. These aspects reflect standard management practices of the facility and the composition of the VHT herd, which consists exclusively of mares. While sex does not appear to be associated with the development or severity of EGUS (Rabuffo et al., 2002; Sykes et al., 2015, 2019; Busechian et al., 2024), findings regarding sex-related differences in HRV are conflicting (von Borrell et al., 2007; Stucke et al., 2015). Indeed, a study reported a higher vagal tone in mares (Clément and Barrey, 1995), while another one did not observe significant sex-related differences (Rietmann et al., 2004b). Consequently, inclusion of both males and females is generally recommended in HRV studies, and the exclusive inclusion of mares may have influenced HRV outcome. Additionally, the sample size was close to the requirement estimated by the a priori power analysis. However, this may have limited the statistical power and the ability to detect smaller effects, particularly given the natural variability of spontaneously occurring gastric ulcers.

Finally, horses in the NT group underwent an additional gastroscopy at T14 to evaluate potential lesion worsening due to the lack of treatment. This procedure, which involved fasting, sedation, and gastroscopy, may have induced acute stress and thereby introduced a potential bias between the T and NT groups. An additional HRV assessment at T14 could have provided further insight into the acute stress induced by fasting, sedation, and endoscopy.

In conclusion, no significant differences in HRV indices were observed following a 4-week treatment for EGUS in horses with naturally occurring gastric ulcers. Although HRV is not routinely applied in equine clinical practice, it represents a valuable non-invasive tool for assessing ANS activity. Unlike previous experimental studies (Louie et al., 2023; Perron et al., 2023), this clinical investigation did not demonstrate a clear association between HRV and EGUS severity or treatment response. The results of the present study should be interpreted carefully, due to the major limitations highlighted. To the authors’ knowledge, this is the first clinical study to assess HRV in relation to naturally occurring gastric ulcers and their treatment. Further studies with longer treatment periods, standardized lesion severity, inclusion of healthy controls, and mixed-sex populations are warranted to better clarify the relationship between HRV, EGUS severity, and therapeutic response.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Funding

This research received no external funding. The research has been completely funded by the University of Pisa.

Author contributions

Conceptualization, D.C., F.B. and M.S.; methodology, F.B., B.S., G.S., A.G. and M.S.; software, G.S.; validation, D.C., F.B. and M.S.; formal analysis, D.C., F.B. and G.S..; investigation, D.C., F.B., B.S., and A.G.; resources, M.S.; data curation, D.C., G.S., F.B., B.S. and A.G.; writing—original draft preparation, D.C.; writing—review and editing, F.B., G.S. and M.S.; visualization, D.C., F.B. and G.S.; supervision, M.S.; project administration, M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.

Data availability

The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request.

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