DISCUSSION

Overview of key findings

This study provides original evidence that tropical environmental conditions attenuate the expected circadian HRV pattern, with only modest nocturnal HR slowing and no clear parasympathetic surge. Furthermore, it links real-time air quality metrics, particularly PM2.5 and AQI, to altered autonomic regulation, characterized by reduced HRV, concurrent decreases in HR, and respiratory cycle changes. These findings suggest a complex and potentially species-specific response that has not previously been described in horses under field conditions.

Unlike previous temperate-climate or exercise-focused HRV studies, the present study highlights the integrated influence of circadian, age-related, and environmental factors on equine autonomic regulation. Circadian variation appeared to be driven more by activity-related modulation than by purely endogenous control, whereas aging was associated with reduced short-term vagal activity and decreased autonomic flexibility.

Blunted circadian autonomic modulation under tropical conditions

Circadian patterns of HR and HRV in this study reflect a dynamic interaction between endogenous rhythms and daily management-related activities. The overall pattern indicates increased autonomic activation during daytime, followed by partial recovery during nighttime. However, the absence of a consistent nocturnal increase in parasympathetic-related indices, including RMSSD, pNN100, and HF, indicates that typical nocturnal vagal predominance was attenuated. Although long-term variability indices, including SDANN and VLF, appeared to increase during evening and nighttime periods, short-term vagal modulation remained relatively unchanged, showing that nocturnal slowing of HR was not accompanied by a proportional increase in beat-to-beat parasympathetic activity. Overall, these findings reflect a blunted circadian autonomic rhythm.

These findings differ from previous studies conducted under temperate conditions, where a clear nocturnal increase in parasympathetic activity and stronger circadian amplitude are typically observed [17, 24]. In contrast, the attenuated nocturnal pattern observed in this study suggests that environmental and management-related factors may interfere with normal autonomic recovery. In particular, daily management routines, including late-afternoon exercise followed by feeding, as outlined in the activity schedule (Table 2), may contribute to transient sympathetic activation extending into the early nighttime period. In addition, environmental conditions characteristic of tropical regions, such as minimal nocturnal cooling and high humidity, may limit physiological recovery during rest [25, 26]. Nocturnal disturbances, including exposure to blood-feeding insects, may further disrupt rest and reduce parasympathetic predominance [16]. However, because sleep quality and nocturnal disturbances were not directly assessed, these mechanisms remain speculative.

Age-associated decline in vagal regulation

Across age groups, the findings provide evidence that aging is associated with decreased parasympathetic regulation and reduced overall autonomic flexibility. This was primarily reflected by declines in short-term HRV indices, including NN, SDNN, RMSSD, pNN100, and HF, together with an increased LF/HF ratio. In contrast, the less pronounced change in long-term variability indices, including SDANN and VLF, suggests that slower regulatory mechanisms may remain relatively intact with aging. Overall, these findings indicate reduced vagal balance or a shift toward sympathetic predominance in older horses.

The findings are consistent with previous studies in horses and humans, which reported an age-related decline in parasympathetic regulation [10, 18, 27–29]. In contrast, the relative stability of long-term HRV parameters observed in the present study may be explained by differences in their underlying physiological regulation. Human studies have shown that short-term indices tend to decline more rapidly with age, whereas long-term indices exhibit a more gradual, continuous decrease [30]. These long-term components are influenced by broader regulatory systems, including neurohumoral control, thermoregulation, and peripheral vasomotor tone, rather than solely by rapid parasympathetic modulation [31, 32]. As a result, long-term variability may appear relatively preserved compared with short-term vagal indices. Although direct evidence in horses remains limited, this framework may help explain the pattern observed in the present study. Importantly, the present findings extend previous equine studies by demonstrating that these age-related autonomic changes occur under tropical environmental conditions, where persistent heat exposure, high humidity, and other environmental stressors may further modulate autonomic regulation.

Environmental modulation of cardiac autonomic function

Environmental factors were associated with cardiac autonomic regulation in horses, reflecting dynamic responses to environmental stressors. Overall, the observed patterns indicate that environmental stressors, particularly air pollution, may disrupt autonomic balance. Higher AQI and PM2.5 were consistently associated with reductions in several HRV indices, including SDNN, RMSSD, pNN100, and HF, together with an increased LF/HF ratio, demonstrating altered autonomic modulation.

Thermal stress is generally associated with increased sympathetic activity [33, 34], and high humidity can exacerbate heat load by impairing evaporative cooling [35, 36]. However, neither temperature nor feels-like temperature showed significant associations with HRV indices in the present study, likely because of the relatively narrow thermal range during the tropical winter period. In contrast, the negative association between humidity and HR observed in this study likely reflects day–night environmental patterns rather than a direct physiological effect of humidity itself, as humidity increased at night when ambient temperature and light intensity declined (Table 3). This interpretation is supported by the observed effects of light intensity, which showed associations consistent with increased arousal and sympathetic activation, leading to elevated HR and shortened NN [37, 38]. The accompanying reduction in SDANN likely reflects a decrease in long-term HRV rather than a specific shift between sympathetic activation and vagal withdrawal [39].

Air pollution-associated autonomic imbalance

Air pollution, particularly PM2.5, was consistently associated with altered autonomic regulation, reflected by reductions in HRV indices alongside negative correlations with HR and respiratory cycle. These findings suggest a complex and potentially species-specific response in horses. Unlike the tachycardia commonly reported in humans and rodents [40–43], horses exhibited reductions in HR and respiratory rate, which may reflect a vagally mediated reflex response. Inhalation of PM2.5 may stimulate pulmonary irritant receptors and C-fibers, triggering reflex pathways that reduce both cardiac and respiratory activity [44, 45]. Despite this apparent vagal activation, the concurrent reduction in HRV indicates disruption of normal autonomic regulation rather than enhanced parasympathetic stability. This dissociation suggests that PM2.5 exposure may induce competing neural effects, including reflex vagal responses alongside impaired autonomic adaptability. In addition, PM2.5 is known to promote systemic inflammation and oxidative stress, which may further compromise autonomic control [40, 44].

Although the impact of PM2.5 on equine health is an emerging field with limited literature, existing studies have primarily documented localized respiratory effects, such as increased pulmonary inflammatory cells and impaired racehorse performance, even at low concentrations of 4–24 µg/m³ [46, 47]. Notably, the mean PM2.5 level in the present study was approximately 53 µg/m³, which was more than twofold higher than those reported in earlier equine research and nearly fourfold higher than the World Health Organization daily threshold of 15 µg/m³ [48]. This concentration is representative of the peak pollution season in Bangkok in January 2025, characterized by prolonged atmospheric stagnation and poor dispersion. This markedly higher exposure may account for the pronounced magnitude of autonomic alterations observed in the study horses. To our knowledge, this is among the first studies to directly examine the relationship between ambient PM2.5 levels and resting HRV parameters in horses under real-world conditions.

Study limitations

This study has several limitations that should be considered. The sample size was relatively small (n = 5 per group), which may limit statistical power. Data were collected during a single seasonal period, the tropical winter in late January, and may not represent conditions across other seasons with greater heat stress. Although management conditions were similar, no direct measurements of physical activity, sleep, nocturnal disturbances such as insect exposure, ammonia, dust, or noise levels were obtained, which may have influenced circadian HRV patterns. HRV was assessed using a Polar H10 sensor rather than a gold-standard electrocardiographic system, and potential measurement limitations under field conditions cannot be excluded. However, the Polar H10 has been widely used in both human and animal HRV research and has demonstrated acceptable accuracy for RR interval detection, particularly under controlled or minimally active conditions [13, 49]. Given that the present study focused on HRV derived from RR intervals rather than detailed electrocardiographic morphology, the use of this device is considered appropriate for the study objectives. Finally, the study population consisted of riding-school horses in an urban tropical setting, which may restrict generalizability to other equine populations, management systems, or regions.

Practical implications for equine welfare

The findings highlight that older horses may be more susceptible to environmental stressors and show clear signs of autonomic dysfunction. Because these changes may intensify under hotter conditions beyond the tropical winter, age-specific management strategies for older horses are warranted. Furthermore, the significant association between PM2.5 exposure and autonomic rigidity underscores air quality as an important welfare concern. Practical interventions, including avoiding exercise during high-pollution periods, reducing physical demands when air quality is poor, improving dust suppression, and monitoring air quality in urban stables, may help support the physiological stability and welfare of horses exposed to these environmental conditions.

Future research directions

Future studies should include larger sample sizes and multi-seasonal data collection to better capture the full range of tropical environmental variability. In addition, continuous monitoring of activity, sleep, and environmental disturbances, including insect exposure and stable conditions, would improve the interpretation of circadian patterns. Finally, controlled experimental studies are needed to clarify causal relationships between air pollution and autonomic regulation in horses.