The findings of this study indicate that oral administration of 8 mg/kg CBD oil influences feline behavior, particularly with respect to sedation, compliance, and temperament. CBD appears to induce sedation in cats, as indicated by significantly increased sedation scores between 2 and 8 h post administration, suggesting a relatively long-lasting sedative effect. The sedation was generally mild, with a median score of 1, typically characterized by staggered walking. However, this effect may have been influenced by stress due to temporary separation from caregivers and exposure to an unfamiliar environment, which may have elevated the baseline anxiety levels of the study cats. Therefore, under less stressful conditions, the degree, onset of action, and duration of sedation might differ. Further studies examining the impact of environmental stress on CBD-induced sedation would enhance understanding of its clinical applicability in cats.

In addition, individual variability was observed, as a 1-year-old domestic shorthair (Cat1) exhibited deeper sedation (scores of 2 to 3) that persisted for up to 12 h post dosing. These findings underscore the importance of accounting for individual differences when determining appropriate CBD dosages for clinical use in cats. In parallel, LC-MS/MS analysis detected specific cannabinoids, including cannabidiolic acid synthase-like 2 (CBDAS3) and tetrahydrocannabinolic acid synthases (THCA, THCA4, THCA5, and THCAS), in plasma samples. Notably, Cat #1 exhibited relatively higher plasma concentrations of THCA and THCA5 during the post-administration period compared to the other cats, coinciding with higher sedation scores and a prolonged duration of effect. This temporal association raises a speculative hypothesis; however, no causal inference can be made regarding the potential contribution of THCA and THCA5 to sedation.

Of note, the detection of cannabidiolic acid synthase-like 2 and THCA synthases—enzymes typically involved in cannabinoid biosynthesis in plants [46]—raises questions regarding their presence in feline plasma following CBD administration. As these enzymes are not endogenously expressed in mammals, their detection likely reflects passive absorption of plant-derived proteins rather than any functional involvement in metabolism. While the biological significance of their presence remains unclear, this exploratory finding underscores the need for continued investigation. Given the complexity of cannabinoid metabolism and the proteomic detection of these enzymes, further research is warranted to determine whether they play any functional role in pharmacokinetics, pharmacodynamics, or cannabinoid efficacy in animals.

The sedative effect of CBD observed in cats in this study is consistent with findings in mice, where CBD exerted sedative and hypnotic effects at doses of 10 mg/kg and 20 mg/kg. These effects were evidenced by increased sleep duration, reduced sleep latency in both normal and insomnia-induced mice, and a significant reduction in locomotor activity at the 20 mg/kg dose compared to controls [47]. In that study, activation of 5-HT_1A receptors was proposed as one of the potential mechanisms, as the sedative–hypnotic effects of CBD were inhibited in mice pre-treated with a 5-HT_1A receptor antagonist [47].

While significant sedation was detected, there were no discernible changes in physiological parameters such as HR, RR, and body temperature throughout the study period. This finding is consistent with previous reports in humans, rats, mice, and horses, which have shown no adverse effects of various CBD concentrations on these physiological parameters [48,49]. These results imply that CBD at the administered dose may produce a sedative effect without causing significant physiological disturbances in healthy cats. This is particularly relevant in veterinary practice, where minimizing adverse effects on cardiovascular and respiratory function is essential when selecting sedation options for animals. Nevertheless, it is important to note that more accurate and reliable methods for assessing cardiovascular and respiratory function, such as blood pressure measurement, electrocardiography, and arterial blood gas analysis, were not utilized in this study. As such, further research remains warranted to thoroughly evaluate the potential side effects of CBD on the cardiovascular and respiratory systems.

A decrease in compliance and temperament scores 2 and 4 h post administration indicated a reduction in the cats’ resistance to handling and assessments. These results may reflect sedative-induced lethargy, as the sedation level increased during the corresponding time periods. Additionally, CBD administration may enhance the emotional well-being of cats by reducing stress and inducing a state of calm, as the anxiolytic properties of CBD have been demonstrated in previous studies. For example, dogs [19] and cats [20] have shown reduced anxiety during separation from caregivers after a 2-week period of daily CBD administration. In mice, CBD at a dose of 3 mg/kg was shown to mitigate stress-induced hyperthermia [50]. These findings support the potential therapeutic application of CBD in managing stress and improving compliance during handling in veterinary patients. In practical terms, such effects may be particularly beneficial during routine procedures that often provoke fear or resistance in animals, such as blood sampling, intramuscular injection, or intravenous catheter placement. The mild sedation observed may also reduce the need for additional restraint or chemical sedation, thereby improving both patient welfare and procedural efficiency. However, species-specific responses, behavioral conditions, and variations in dose regimens must be considered. As an example, CBD treatment in shelter dogs with behavioral disorders did not lead to behavioral improvement and was still associated with a significant increase in hair cortisol levels following exposure to common stressors [51]. These contrasting findings highlight the complex and context-dependent effects of CBD across different species and physiological states. According to plasma cannabinoid analysis, although the levels of detected THCA synthases and CBDAS3 did not significantly differ across timepoints, most exhibited a slight increasing trend after 2 h compared to 1 h post administration. While this trend was temporally associated with notable changes in compliance, temperament, and sedation scores, further investigation with a larger sample size is needed to clarify any potential association between these cannabinoids and behavioral effects in cats.

Interestingly, the observed time course of significant changes in the level of sedation, compliance, and temperament aligns with previously published pharmacokinetic data, which indicate that peak CBD plasma concentrations occur between 2 and 2.6 h following the administration of various doses, such as 1.37, 2, 2.5, 5, 10, 20, and 40 mg/kg of CBD in cats [23,25,26,52]. This information can be useful in guiding both owners and veterinary staff to wait for 2 h after CBD administration to ensure that the drug reaches its maximum effect, thereby increasing the likelihood of successful clinical interactions with stressed cats.

A single dose of 8 mg/kg CBD did not result in significant changes in mechanical sensitivity and nociceptive thresholds in cats, as both von Frey filament and algometer testing produced results consistent with baseline measurements across all post-administration timepoints. These findings diverge from prior studies that indicated antinociceptive and analgesic properties of CBD in animal models. In canine osteoarthritis studies, administration of 2 mg/kg CBD twice daily in various breeds and 50 mg/day in large domestic dogs reduced pain scores, increased comfort and activity levels, and improved overall quality of life [17,18]. In a Parkinson’s disease model, parkinsonism-induced mice exhibited reduced thermal thresholds in hot plate and tail flick tests and lower mechanical nociceptive thresholds in von Frey filament tests, while intraperitoneal CBD at doses of 10 and 100 mg/kg increased these thresholds, alleviating hyperalgesia and allodynia [53]. In a neuropathic pain rat model, 3 mg/kg of intraperitoneal CBD effectively reversed mechanical and thermal allodynia, hyperalgesia, and associated anxiety-like behaviors [54]. Moreover, in a rat arthritis model, transdermal CBD at doses of 6.2 and 62 mg/day significantly reduced joint swelling and spontaneous pain scores, while restoring paw withdrawal latency to near-baseline levels in response to noxious heat stimulation [55].

The absence of antinociceptive effects observed in the present study may be attributed to the context-dependent nature of CBD’s efficacy in pain modulation. Factors such as the type of pain (e.g., nociceptive, acute, chronic, inflammatory, neuropathic pain), each with distinct underlying mechanisms, along with dosage regimens and interspecies differences, may contribute to this variability. It is possible that the use of healthy cats without an induced pain condition in this study may not have activated the relevant pain pathways that CBD is more likely to modulate. This observation is compatible with the known pathophysiological roles of cannabinoid receptors in neurological and inflammatory diseases [7], as well as CBD’s interactions with non-cannabinoid receptors, such as 5-HT_1A and TRPV1, which are particularly implicated in inflammatory states [8,9,10,11,12]. Consequently, CBD’s antinociceptive effects may be more pronounced in pathological pain conditions, contrasting with its limited impact under normal physiological states.

Another important consideration is the duration of CBD exposure and the potential for delayed or cumulative effects, as observed in studies on canine osteoarthritis, where analgesic outcomes were reported after 2 and 4 weeks of daily treatment [17,18]. This contrasts with the present study, which assessed effects following a single dose. Longer-term use or higher dosing (e.g., greater than 8 mg/kg) may yield different outcomes. Therefore, exploring extended treatment protocols or alternative regimens could provide deeper insights into CBD’s potential for pain modulation in cats. In addition, interspecies differences in cannabinoid receptor distribution may contribute to the limited antinociceptive response observed in cats. For instance, dogs have been shown to possess a significantly higher number of CB1 receptors in hindbrain structures compared to humans [4], suggesting that receptor localization and density could influence the pharmacodynamic effects of cannabinoids across species. While comparable data for cats remain limited, such anatomical differences may partially explain the variability in analgesic efficacy observed between feline and non-feline models.

Furthermore, certain constraints should be considered when interpreting the mechanical testing results. One and two cats were excluded from sensitivity and threshold testing, respectively, due to unexpected responses. These exclusions may have reduced the statistical power and limited the ability to detect potential treatment effects on mechanical sensitivity and nociceptive thresholds. One cat exhibited a complete absence of response to von Frey filament testing at all timepoints, possibly due to fear-induced immobility or hyposensitivity stemming from an undetected condition during the initial health screening. The other two displayed strong aversive reactions to the algometer upon contact. These reactions underscore differences in sensitivity to mechanical stimuli among cats. This variation may have been influenced by the diverse backgrounds of the cats, as they were sourced from different owners and had no prior exposure to or familiarity with experimental equipment or environments. Such unfamiliarity may have contributed to fear, hyperexcitement, or hyperactivity. To minimize individual disparities in mechanical sensitivity and nociceptive threshold testing, future studies could benefit from using cats raised in a standardized experimental setting, with more uniform characteristics such as age and breed. These findings also highlight the limited feasibility of using certain nociceptive assessment tools under conditions where animals are unfamiliar with the testing setup. Further refinement and validation of these methods are necessary to improve their applicability in feline studies.

Previously reported adverse effects of CBD oil in cats include drowsiness, lethargy, excessive salivation, and elevated liver enzymes [23,24,25,26,27]. In the present study, hypersalivation, emesis, drowsiness, and erythema of the ear pinnae were observed, emphasizing the need for careful consideration when prescribing CBD oil for feline patients. A transient elevation in AST above the reference range was detected 24 h post administration but returned to baseline by two weeks after treatment. The observed increase may be associated with the liver’s metabolic processing of CBD via cytochrome P450 enzymes, indicating elevated hepatic activity rather than actual liver damage [56]. However, caution is advised when using CBD oil in cats with pre-existing hepatic conditions. Hypersalivation occurred immediately after administration in 44% of cats, possibly due to the taste of the capsule or leakage from a broken capsule. Vomiting occurred as a single event in two cats; no diarrhea or soft stool was reported. The episodes of vomiting may be attributed to individual sensitivity to the taste or odor of the CBD oil, irritation from the capsule contents if rupture occurred orally, or gastrointestinal intolerance to the carrier oil. While the CBD–THC ratio of ≥20:1 minimizes psychoactive effects, trace THC content may also contribute to nausea or emesis in susceptible individuals [56]. Such reactions underscore the importance of formulation palatability and delivery method in feline patients. Drowsiness, observed in 89% of cats, was the most prominent effect and may be related to the sedative–hypnotic properties of CBD. Erythema of the ear pinnae and increased water intake were noted in one cat, potentially indicating hypersensitivity or an allergic reaction to the CBD oil. However, it remains unclear whether the reaction was triggered by the CBD itself or by the carrier oil used as a vehicle. Although these adverse effects were generally mild and transient, they raise potential concerns about the tolerability of the 8 mg/kg dose used. Careful dose titration and monitoring are therefore necessary to minimize the risk of undesirable side effects.

These findings underscore the need for careful consideration when prescribing CBD oil for feline patients, particularly in light of potential adverse effects such as emesis, hypersalivation, and elevated AST levels. Given the increasing accessibility of CBD products for pets and the potential for misuse or misinformed application, it is essential to communicate evidence-based information to the broader public. Social media may serve as an effective tool in this regard; for example, a recent study demonstrated how platforms such as Instagram can help convey complex scientific topics—such as safety concerns and the behavioral and physiological effects of CBD—to a wider audience [57].

Limitations of this study should be acknowledged. First, the scaling systems used to assess sedation, temperament, and compliance in cats were not formally validated prior to use, and interpretations were not guided by veterinary behavior specialists. However, these systems have been employed in previous studies investigating the effects of opioids and other central nervous system depressants in this species [32,33,34,35], and consistency was maintained by using a single trained evaluator throughout. Future studies should incorporate expert input and formal validation to enhance the reliability of behavioral assessments. Second, the temperament score combined signs of fear and aggression into a single ordinal scale, which may have masked important distinctions in the cats’ behavioral responses to CBD. Future studies should consider separating these emotional responses to better elucidate the specific effects of CBD on feline behavior. Third, the cats were not acclimated to the study environment or equipment before testing began. This approach aimed to establish a consistent baseline by ensuring that all cats—despite originating from diverse household backgrounds—were housed overnight in the same novel environment and separated from their owners prior to the study. This was intended to standardize initial conditions while still reflecting the variability typically seen in real-world clinical populations. However, the lack of acclimatization may have contributed to unexpected responses during mechanical testing, as previously discussed, and some of the observed behavioral responses may have reflected environmental stress rather than pharmacological effects alone. Fourth, the preliminary dose-finding data were derived from a small number of animals. While useful for estimating the CBD dose, the limited sample size reduces the strength and generalizability of these findings. This issue extended into the main study, where financial constraints also led to a modest sample size and the absence of a control group. These limitations reduce the generalizability of the findings and warrant cautious interpretation. In the absence of a placebo group, the potential influence of other factors—such as environmental stress and circadian or repeated handling influences—on behavior and sedation cannot be ruled out. Future studies employing randomized, placebo-controlled designs are necessary to validate the behavioral and physiological effects observed in this investigation.

Another limitation of this study lies in the absence of global multiple comparison correction across all endpoints. This raises the potential risk of inflated Type I error due to the large number of comparisons. However, given the exploratory nature of this study and the fact that these outcomes represent distinct biological domains evaluated independently, a within-family correction (e.g., Benjamini–Hochberg) was applied within each outcome domain to balance Type I error control with statistical sensitivity. Applying a more conservative global adjustment (e.g., Bonferroni across all comparisons) would likely have increased the risk of Type II error, reducing the ability to detect potentially meaningful effects in this exploratory, multi-domain investigation [58,59]. Nonetheless, this methodological choice should be acknowledged when interpreting the significance of individual findings. Moreover, although the CBD product used in this study contained only trace amounts of THC (CBD–THC ≥ 20:1), the possibility that these minimal levels contributed to the observed effects or adverse outcomes cannot be entirely excluded. This consideration is particularly relevant given the plasma detection of THCA and THCAS, which may suggest some degree of biological activity even at low concentrations. Additionally, the limited number of plasma sampling timepoints may have hindered the detection of more detailed pharmacokinetic patterns. Future studies should incorporate more frequent sampling and larger sample sizes, both to validate the current findings and to facilitate the identification of cannabis-related proteins potentially involved in behavioral modulation, which could offer valuable insights for therapeutic development.