In the present study, oral administration of CBD at 2 mg/kg twice daily for 21 days did not significantly affect body weight, BCS, CNS, or blood triglyceride concentrations, but was associated with an increased insulin response during the OST.

EMS, which shares pathophysiological similarities with MetS, is characterized by insulin dysregulation, impaired glucose tolerance, insulin resistance, and dyslipidaemia, including hypertriglyceridaemia (16). Similarly, MetS is a multifactorial and modifiable condition associated with increased risk of cardiovascular disease, type 2 diabetes, and other adverse health outcomes, driven by mechanisms such as insulin resistance, adipose tissue dysfunction, systemic inflammation, and dyslipidaemia (45).

Preclinical studies indicate that CBD exerts beneficial effects on body weight and food intake regulation, glucose and lipid metabolism, insulin resistance, neuroinflammation, and obesity-related behavioural disturbances (46–54). Although the mechanisms of CBD are not yet fully understood, several central and peripheral targets have been proposed based on preclinical and in vitro studies in the context of obesity and metabolic syndrome. For example, CBD has been suggested to interact with cannabinoid (CB1, CB2) and serotonin (5-HT1A) receptors, which may contribute to effects on appetite regulation and reward-related feeding behaviour (46). Peripherally, CBD may improve insulin sensitivity and lipid metabolism through cannabinoid receptor modulation and activation of peroxisome proliferator-activated receptor gamma (PPARγ), potentially promoting adipose tissue browning, enhancing glucose utilization, and reducing dyslipidaemia. Additionally, CBD has been reported to exert anti-inflammatory effects and may lower circulating lipid levels via regulation of low-density lipoprotein metabolism. Together, these interconnected mechanisms may support the therapeutic potential of cannabidiol in managing obesity and related metabolic disturbances (46).

In the current study, body weight and BCS remained stable during the treatment period, and the observed difference in BCS between groups is likely attributable to baseline variability rather than a treatment effect. CNS scores showed only a marginal decrease following treatment. In contrast, in a mouse model of high-fat diet–induced obesity, longer-term CBD administration at a higher dose (25 mg/kg for 9 weeks) reduced weight gain, lowered serum glucose, and decreased white adipose tissue mass and adipocyte size (55). In the ponies included in this trial, TG levels remained stable in controls and showed a non-significant increase in treated ponies. Blood glucose concentrations during OST largely followed the expected post-prandial pattern in all groups. Insulin responses during the OST increased in CBD-treated ponies, while responses in control animals remained stable. The observed increase following CBD administration should be interpreted within the context of a longitudinal, within-subject effect. Although baseline insulin levels were higher in the CBD-treated group, the stability of insulin responses in control animals across both OSTs supports a treatment-associated change rather than random variability. Nevertheless, residual baseline differences cannot be entirely excluded, and confirmation in independent cohorts is warranted.

Diet composition is known to significantly influence metabolic responses and gut homeostasis in horses, with starch-rich conditions promoting dysbiosis and inflammatory pathways that may affect glucose–insulin dynamics (56). More generally, nutritional factors, particularly forage quality and dietary composition, play a crucial role in modulating metabolic health and digestive function in horses (57), while feeding behaviour and time-activity patterns are closely linked to metabolic regulation and overall physiological balance (58). Importantly, in the present study, feeding conditions and diet composition were consistent across all animals; however, differences in insulin response were observed only in the CBD-treated group, suggesting that the observed effect is unlikely to be attributable to dietary variation alone. Nevertheless, the study design does not allow differentiation between a potential worsening of insulin dysregulation and a modulation of insulin secretion dynamics, and therefore this finding should be interpreted with caution.

The observed effects of CBD may vary depending on multiple contributing factors, including species-specific receptor distribution and the targeted clinical outcome. Cannabinoid receptor localization and ligand binding differ across species, indicating that pharmacological responses may not be directly comparable (59, 60). In addition, CBD exerts dose-dependent effects via multiple cellular targets, with distinct plasma concentrations required for different clinical outcomes; such actions, mediated by receptor activation, antagonism, or inhibition, have been previously described (61).

Rafailovska et al. (62) demonstrated that the metabolic effects of CBD are both dose and route-dependent. In healthy rats, intragastric CBD improved glucose regulation only at the highest dose tested (50 mg/kg), whereas lower doses (0.5 and 5 mg/kg) were ineffective, and intraperitoneal administration produced no significant glycemic effects. In diabetic rats treated intragastrically for 8 days, CBD exerted dose-specific metabolic effects: 25 mg/kg reduced blood glucose and increased insulin levels, while the most pronounced metabolic improvements were observed at 50 mg/kg, including the greatest reduction in glucose concentrations, a moderate rise in insulin, suppression of gluconeogenic enzymes, and improvements in lipid parameters. In contrast, the highest dose (100 mg/kg) showed minimal metabolic benefit. Overall, these findings emphasize the critical role of both dose and administration route in determining CBD’s metabolic efficacy.

It is possible that higher doses of cannabidiol attenuate its effects on glycaemic control through receptor desensitization, activation of counter-regulatory mechanisms, or non-specific interactions with additional molecular targets that influence metabolic pathways (62).

While increases in insulin concentrations could raise concerns in EMS-affected horses, particularly with respect to laminitis risk, existing experimental evidence indicates that CBD may improve metabolic parameters at certain doses, even in the presence of moderate insulin elevation. This suggests that the metabolic effects of CBD may depend on a balance between dose, metabolic status, and species, highlighting the need for further targeted studies. From a clinical perspective, these findings suggest that CBD use in EMS-prone horses or ponies should be approached with caution, particularly with regard to dose selection and metabolic status, until more robust evidence becomes available. Although CBD was well tolerated and did not adversely affect most metabolic parameters, the potential for enhanced post-prandial insulin response may be clinically relevant, given the established link between hyperinsulinemia and laminitis risk. Therefore, until further evidence is available, the use of CBD in EMS-prone individuals should be approached cautiously and under close veterinary supervision. In horses, positive clinical effects on mechanical allodynia and crib-biting have been reported at relatively low oral doses of cannabidiol (0.5 mg/kg twice daily), despite the typically low systemic cannabidiol concentrations observed at these doses in the literature (10, 11).

Collectively, these findings indicate that varying plasma cannabidiol concentrations can be clinically relevant and emphasize the need for dose-escalation studies with integrated pharmacokinetic analyses and efficacy assessments across multiple health conditions to more precisely define therapeutic exposure ranges. Future studies should also investigate longer-term administration protocols and dose–response relationships, with particular emphasis on insulin dynamics in EMS-affected horses, and consider the application of advanced monitoring approaches to better characterize metabolic responses under field conditions.

In the present study, oral CBD administration was well tolerated in all horses, with no adverse effects or changes in appetite observed throughout the study. Physiological parameters remained within normal ranges, indicating a favorable safety profile. Mean heart rate was within physiological limits in all animals. Although minor group and sex-related variations were observed, these differences were small in magnitude and are unlikely to be clinically relevant with respect to the primary metabolic outcomes of the study. These findings should nevertheless be interpreted with caution given the limited sample size.

Biochemical analyses revealed minor changes in ALT, glucose, fructosamine, total bilirubin, and triglycerides over the 21-day trial. ALT levels showed slight elevations in all horses, independent of treatment. Blood glucose and triglycerides were elevated in several ponies, particularly those with equine metabolic syndrome, with changes occurring regardless of CBD administration. Fructosamine levels, which were elevated in some ponies at baseline, normalized by the end of the trial and were not influenced by CBD treatment. Total bilirubin increased slightly in both groups but remained within normal limits, with no evidence of CBD-related effects. Overall, these findings indicate that chronic CBD administration did not adversely affect key biochemical parameters.

Evidence from other species highlights the potential for dose-dependent liver effects of CBD. In humans, therapeutic CBD (1,500 mg/day) caused ALT elevations in some healthy adults, and Ewing et al. (63) demonstrated dose-dependent liver injury in mice at doses above 50 mg/kg. In contrast, studies in horses suggest that modest, long-term oral CBD administration is well tolerated. Wang et al. (64) observed normal serum liver enzymes and unremarkable hepatic biopsies in horses treated with 1 mg/kg twice daily for 6 weeks. Similarly, Yocom et al. (28) reported transient, non-dose-dependent elevations in liver enzymes (GGT, AST, sorbitol dehydrogenase) in horses administered 0.5 or 1.5 mg/kg CBD orally twice daily for 6 weeks, which returned to reference intervals within 10 days. In the authors’ previous study, only slight, clinically irrelevant increases in GGT, total bilirubin, and LDH were observed (30). These results suggest that modest, long-term doses of these cannabinoids appear safe in horses, though further studies are needed to evaluate the effects of higher doses or prolonged administration on liver function and overall drug efficacy.

Beyond liver safety, ACTH concentrations increased in all eight elderly horses over the study period independent of treatment. A comparable pattern was observed for blood glucose, which increased in most ponies by the end of the trial regardless of treatment or EMS status. Previous studies have shown that even short-term stressors, such as road transport, can induce elevations in circulating ACTH and glucose concentrations in horses (65, 66). The generalized nature of these elevations, including in non-EMS animals, supports the interpretation that stress associated with the experimental conditions contributed to the observed changes rather than a direct metabolic effect of CBD.

The second aim of the present study was to characterize the plasma pharmacokinetics of CBD and its two main metabolites (7-OH-CBD and 7-COOH-CBD). Repeated oral administration of CBD at 2 mg/kg every 12 h resulted in an increase in peak plasma concentrations from the first to the final dose, consistent with the observed accumulation ratio (Table 1). Specifically, after administration of the first dose, the C_max of CBD was 22.789 ± 13.235 ng/mL, whereas by the end of the study, following the final dose, it had increased to 39.933 ± 14.448 ng/mL. Overall maximal plasma concentrations were consistent with those reported in the existing equine literature, while variability among studies likely reflects differences in dose, formulation, route of administration, and dosing regimen, as well as individual animal characteristics (3).

Oral administration has consistently been associated with dose-dependent increases in C_max (29). For example, Thomson et al. (67) reported peak plasma concentrations of approximately 40 ng/mL following administration of 8 mg/kg CBD via nasogastric tube, while Sánchez de Medina et al. (68) observed higher C_max values after oral administration of 10 mg/kg CBD, particularly when using oil-based and micellar formulations. The markedly higher plasma concentrations achieved with micellar preparations suggest that formulation plays a critical role in enhancing CBD bioavailability. In addition, alternative administration routes that bypass first-pass hepatic metabolism, such as transmucosal delivery, have been shown to yield measurable plasma concentrations even at low doses (32). Feeding status may further influence oral absorption, as demonstrated in humans; however, in horses, a crossover study comparing oil and paste formulations administered under fed and fasted conditions did not identify significant differences in pharmacokinetic parameters, although the paste formulation achieved peak plasma concentrations more rapidly, suggesting formulation-dependent differences in absorption kinetics (69). Repeated dosing has also been associated with higher peak concentrations, potentially reflecting drug accumulation and improved absorption over time, as reported by Williams et al. (29) following daily administration of full-spectrum CBD pellets.

Time to peak concentration remained relatively stable, and steady-state plasma levels were reached after approximately 4–5 days of dosing, as indicated by consistent pre-dose concentrations. These results demonstrate predictable pharmacokinetics and support the feasibility of twice-daily oral dosing in ponies.

Williams et al. (29) administered CBD orally to horses at 2.0 mg/kg once daily for 7 days. Plasma CBD concentrations increased over the initial dosing period and reached steady-state levels after approximately 3 days, with a mean C_max of approximately 51 ng/mL occurring at a T_max of about 2.4 h post-dose and a terminal half-life of about 10 h.

Sánchez de Medina et al. (68) conducted simulations of different CBD formulations and dosing regimens, using steady-state concentrations (after the 8th day) to calculate the probability of target attainment (PTA) for plasma levels ranging from 1 to 100 ng/mL. The simulations indicated that micellar formulations produced higher C_max and shorter t_max, whereas oil formulations exhibited lower C_max but more stable concentrations at 24 h. Oral CBD at 10 mg/kg every 12 h was predicted to maintain plasma levels up to 40 ng/mL at steady state, while once-daily dosing achieved levels above 10 ng/mL throughout the dosing interval. However, these findings are based on simulations, and without clinical validation they remain predictive only.

In a multiple-dose study in horses, oral administration of CBD paste at 3 mg/kg twice daily for 15 days (n = 6) resulted in steady-state plasma concentrations being reached by Day 2 (20).

In a human multiple-dose study (n = 43), participants received an oil formulation at total daily CBD doses of 120, 240, 360, or 480 mg (with proportionally low THC content), administered orally in divided doses every 12 h for 7 consecutive days. Steady-state plasma CBD concentrations were achieved by Day 7, with higher CBD doses producing proportionally higher plasma CBD concentrations (70).

The t_max observed in the present study (Table 1) was consistent with most previous reports, which describe values ranging from 1 to 5 h (3).

In the present study, the t_½ of CBD in horses was approximately 5.5 h after the first dose and 4 h after the final dose (Table 1). These values are shorter than those reported in most previous equine studies (10–12 h) (28, 29). In contrast, Eichler et al. (20) reported a markedly longer t_½ of approximately 161 h following multiple-dose administration of CBD paste (3 mg/kg twice daily for 15 days). Conversely, Wang et al. (64) observed a substantially shorter elimination half-life, comparable to that reported in the present study, of approximately 4 h following oral CBD administration at 1 mg/kg twice daily for 6 weeks. In humans, the reported t_½ was 2–5 days after chronic oral administration (28, 29, 71), while in dogs, t_½ of orally administered cannabidiol vary widely across published pharmacokinetic studies, ranging from approximately 1 to 19 h (72).

Such variability in pharmacokinetic parameters across species, studies, and even individuals has been described following oral administration of cannabidiol formulations, even when doses are normalized. This variability may reflect small sample sizes, differences in sampling schedules, and interindividual factors such as breed, age, and sex. Age-related physiological changes affecting hepatic and renal function may further influence drug disposition, as may sex-related differences in metabolism. In addition, cannabidiol formulation and composition, whether purified or combined with other phytocannabinoids, may contribute to the observed pharmacokinetic variability (72).

Cannabinoid metabolism occurs primarily in the liver, where CBD is converted by cytochrome P450 enzymes to the active metabolite 7-OH-CBD and subsequently to the inactive metabolite 7-COOH-CBD (3). The latter persists longer in the body and is therefore considered a useful biomarker of cannabinoid exposure. CBD also undergoes direct glucuronidation via UDP-glucuronosyltransferases (73). Elimination occurs predominantly via feces, with a smaller proportion excreted in urine, mainly as unchanged or glucuronidated CBD (73, 74). Cannabinoid metabolic pathways vary considerably between species (25), and the specific metabolic profile of CBD in horses remains incompletely characterized. In humans, oral CBD undergoes extensive first-pass metabolism, resulting in rapid formation of metabolites (75, 76).

In the present study, repeated oral administration of CBD in ponies revealed a prolonged persistence of both the parent compound and its primary metabolite (7-COOH-CBD) in plasma following cessation of treatment. Cannabidiol remained detectable for up to 35 days after the final dose in most ponies, while the 7-COOH-CBD persisted in all animals through the final sampling time point. This extended detectability suggests slow elimination and possible metabolite accumulation in horses. These findings highlight the importance of considering extended metabolite persistence and elimination times when evaluating the safety, interpretation of plasma concentrations, and clinical use of CBD in equine practice.

Most international equine sport authorities prohibit all natural and synthetic cannabinoids; however, in 2022 the Fédération Equestre Internationale (FEI) reclassified CBD and its precursor cannabidiolic acid (CBDA) as controlled medications rather than prohibited substances (20). This regulatory shift highlights the need for a clear understanding of CBD pharmacokinetics in horses, as detection may still result in an adverse analytical finding but is evaluated under different rules that acknowledge potential inadvertent exposure.

The primary limitation of the present study was the small sample size (n = 13). Nevertheless, it should be noted that previous investigations addressing similar research questions have employed comparable sample sizes (9, 18, 19, 24, 25, 29, 30, 64, 68, 69). The consistency of the present results with those reported in the existing literature supports the adequacy of the sample size within this research context. Although stratified randomization was applied to balance key characteristics between groups, the relatively small sample size may have limited its effectiveness, and residual imbalance between groups cannot be excluded. In addition, it should also be acknowledged that clinical outcomes in equine studies are often influenced by multiple interacting physiological and environmental factors, reflecting the inherent biological variability of this species (77). Additional limitations of the present study include the exclusive analysis of blood samples, as urine samples were not collected; the relatively short treatment duration and fixed dosing regimen; and the lack of concurrent structured management interventions such as dietary restriction or exercise modification, which may also have influenced the observed outcomes.