Steroid Hormone Changes after Short-Term High-Dose Exposure to Cannabidiol in Healthy Subjects: An Exploratory Study

Abstract

Introduction

Cannabis-derived cannabidiol (CBD; Epidiolex®) is FDA-approved for seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex, yet its effects on human steroid hormone regulation remain unclear. This study evaluated changes in five plasma steroid hormones and one precursor bile acid after short-term high-dose oral CBD administration in healthy subjects.

Methods

Twelve participants (6 males, 6 females) completed a randomized, placebo-controlled crossover study and received oral CBD 750 mg twice daily for 3.5 days or placebo, separated by a washout period. All subjects received methylphenidate on the final clinical day for an additional independent study objective. Plasma samples collected after the final dose were analyzed by LC-MS/MS. Statistical analyses assessed CBD-associated hormonal changes and correlations with CBD and metabolite concentrations. This study was supported in part by the State of Florida Consortium for Medical Marijuana Clinical Outcomes Research.

Results

A significant decrease in the precursor steroid 21-OH-progesterone was observed in the CBD group compared with the placebo group. Cortisol and corticosterone levels increased consistently across all CBD-administered participants, and testosterone levels rose significantly in male subjects. No significant differences were identified between the CBD and placebo groups in cholic acid concentrations. All measured hormone levels remained within physiological ranges.

Conclusions

This study provides novel evidence that CBD modulates key components of the steroidogenic and endocrine systems, revealing previously unappreciated endocrine effects in humans.

Introduction

Epidiolex^® is the first FDA-approved pharmaceutical drug to contain a purified form of cannabidiol (CBD) derived from the cannabis plant. The labeled indications are for the treatment of seizures associated with Lennox-Gastaut syndrome and Dravet syndrome [1]. Moreover, CBD is a cannabinoid that is widely used as an over-the-counter supplement and a component of medical cannabis [2]. The metabolism of CBD in humans has been published previously [3]. CBD is extensively metabolized to its major active metabolite, 7-hydroxy-CBD, primarily by the cytochrome P450 enzymes CYP2C19 and CYP2C9 [4, 5]. 7-hydroxy-CBD showed comparable pharmacological activity to the parent compound [5] and is subsequently metabolized to 7-carboxy-CBD by CYP3A4 [6].

The pharmacology of CBD remains largely unexplored. Clinically, the primary therapeutic effects of CBD include sedative and anticonvulsant properties [7]. CBD’s central nervous system pharmacology is largely attributed to its function as a negative allosteric modulator of the type 1 cannabinoid (CB1) receptor [8]. When activated by the endogenous cannabinoids, CB1 suppresses neuronal activity [9]. CBD, as a negative allosteric modulator, binds to the CB1 receptor and induces a structural change that decreases the receptor’s binding affinity and signaling efficacy of the located neurons [8].

Numerous studies have investigated the biological role of CB1 receptors in steroid regulation [10]. CB1 is widely distributed on GABAergic and glutamatergic neurons in the hypothalamus, a key regulatory center for endocrine function [11]. The hypothalamic-pituitary-adrenocortical (HPA) axis primarily regulates circulating glucocorticoids [10, 12, 13], while the hypothalamic-pituitary-gonadal (HPG) axis regulates reproductive and fertility-related hormones [14]. In vitro studies indicate that CBD may influence both male and female reproductive systems by modulating sex steroid hormone production [15, 16]. In addition to its central expression, CB1 receptors are also present in the adrenal cortex [13] and peripheral neurons [17], suggesting a broader role in neuroendocrine regulation. In the study by Zuardi and colleagues [18], the effects of higher doses of CBD (300 mg and 600 mg), relative to placebo, on plasma prolactin, growth hormone, and cortisol concentrations were evaluated. These investigators reported that CBD exposure attenuated the expected circadian decrease in cortisol concentrations. Although the sample size was limited, the findings suggested a potential dose-dependent effect of CBD on these endocrine parameters.

Under normal physiological conditions, steroid hormone concentrations exhibit significant daily and interindividual variability. Moreover, many steroid hormones differ significantly between males and females. Table 1 presents the reference ranges for the steroids analyzed in this study, highlighting sex-specific differences as well as hormonal fluctuations across the various phases of the female menstrual cycle [19].

Table 1.

Reference hormone concentrations

​	Significant sex difference?	​	Lower bound, ng/mL	Upper bound, ng/mL
21-OH-progesterone	No	​	0.00	0.16
Corticosterone	No	​	0.59	14.29
Cortisol	Yes	Male	48.57	233.43
		Female	43.86	253.73
Progesterone	Yes	Male	0.27	0.90
		Female	0.02	17.24
Testosterone	Yes	Male	2.19	10.70
		Female	0.09	0.66
Cholic acid	No	​	Not reported	2,043

The reference ranges were established in 298 healthy subjects, and sex differences were determined using statistically significant t tests.

A comprehensive clinical investigation into the effects of CBD on circulating steroid hormones has not yet been conducted, highlighting a significant gap in the current research area. The present study investigated changes in plasma steroid hormone concentrations following short-term, high-dose oral CBD administration in 12 healthy subjects. All subjects received methylphenidate (MPH) on the final clinical day for an additional independent study objective. A total of 5 steroid hormones and one bile acid were measured across two study arms. These included two glucocorticoids (cortisol and corticosterone), two reproductive steroids (testosterone and progesterone), and two precursors (21-OH-progesterone and cholic acid). The findings have the potential to enhance our understanding of the pharmacology, side effects, and clinical implications of CBD and medical cannabis administration.

Methods

Study Design Overview

Plasma samples were sourced from a previously conducted CBD drug interaction study involving unmedicated healthy volunteers [20]: in brief, a randomized, crossover design (n = 12; 6 males and 6 females). Participants received either an oral CBD solution or a placebo solution. The dosing regimen included 750 mg administered twice daily for three consecutive days, followed by a single 750 mg dose on the morning (8 a.m.) of the fourth day. Plasma samples were collected after 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h immediately before (0 h) and after the final dose of CBD. Collected samples were placed on ice for processing, and plasma was stored at −70°C until analysis. As part of the drug interaction assessment, 10 mg of MPH was administered to subjects on the day of sample collection. Other information can be found in the original publication [20].

The FDA-approved Epidiolex^® formulation (100 mg/mL CBD solution) of CBD was used in this study. Greenwich Biosciences Inc. (Carlsbad, CA, USA) generously provided Epidiolex^® as well as a matching placebo solution in support of this investigation. Study participants were twelve healthy volunteers (6 men and 6 women) aged 21–44 years (26.7 ± 6.5 years; weight, 65.8 ± 14.1 kg; mean ± SD) who completed the study protocol. Subjects were determined to be healthy by medical history, physical examination, and routine laboratory assessments, including serum electrolytes, liver function tests, urinalysis, and complete blood count. Apart from oral contraceptives, the use of prescription or over-the-counter medications was not permitted. These restrictions also apply to nutritional supplements, vitamins, and energy drinks. To prevent confounding from external cannabinoid exposure, all participants underwent urine THC screening to exclude cannabis use outside the study protocol. Subjects were provided with a standardized breakfast (bagel, cream cheese, and orange juice), which was consumed over approximately 30 min prior to dosing with CBD or placebo. All participants tolerated the CBD dosing well, and no adverse effects were reported.

Plasma CBD concentrations and metabolites were measured in these subjects, and these results are reported in a separate publication [20] and also in the online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000552140). The correlation between CBD pharmacokinetics and hormonal effects is presented in the online supplementary material. Given the small sample size, this correlation is not discussed further in the main manuscript.

Bioanalysis

Plasma samples were shipped at −20°C and analyzed by Callen Analytical Service (Boston, MA, USA). All equipment and analytical standards were purchased and provided by the company. In brief, 100 µL of plasma was added to 96-well plate. And 10 µL of an internal standard (ISTD) solution consisting of 50:50 water:methanol containing mifepristone (100 ng/mL) was added. Also, 75 µL of saturated ammonium sulfate was aliquoted into all wells, and the mixture was pipetted up and down several times. The plate was then capped and vortexed for 5 min. Subsequently, 150 µL of acetonitrile was added to each well, followed by capping and vortexing for 10 min, and centrifugation at 4,000 rpm for 10 min Then, 120 µL of the supernatant was collected and injected into the LC-MS for analysis. The lower limit of quantitation for cholic acid is 1.00 ng/mL, for 21-OH-progesterone is 0.2 ng/mL, for progesterone is 0.1 ng/mL, for testosterone is 1 ng/mL, for cortisol is 2 ng/mL, and for corticosterone is 0.1 ng/mL. Specific instrument parameters are provided in the online supplementary material.

Statistical Analysis Approach

Illustrations were made by BioRender (Toronto, ON, Canada). Statistical Analysis System^® (SAS, Cary, NC) 9 and GraphPad PRISM (Boston, MA, USA) were used for analysis. A paired t test was conducted for each hormone with the CBD and placebo groups, paired within subject. For the hormones reported to have sex differences, the analysis was conducted separately in males and females (testosterone and cortisol, Table 1). Each data point represented the mean plasma concentration of 9 time points for each subject. To further investigate the biological role of CBD, further analysis has been done for all hormones showing significant changes with CBD area under the curve (AUC) to assess the dose-dependent effects of CBD. Linear regression was performed on the change in hormone concentrations relative to the AUC of CBD.

Results

Cholic Acid

No significant differences in cholic acid concentrations were observed between the CBD and placebo groups (Fig. 1; Table 2). Plasma concentrations in both groups remained within the normal physiological range for cholic acid (<2.04 μg/mL).

Fig. 1.

Plasma concentrations of cholic acid. Left: Plasma cholic acid concentrations over time following the final CBD administration. Each point represents the mean of all subjects, and error bars indicate the standard deviation. Right: Paired t test comparing mean cholic acid concentrations between the CBD and placebo groups. Each point represents the average hormone concentration of an individual subject. Physiological concentration of cholic acid is not higher than 2,043 ng/mL.

Table 2.

Statistical results for the effects of CBD on steroid hormones

​	Cholic acid		21-OH-progesterone		Testosterone (male)		Cortisol (male)		Cortisol (female)		Corticosterone
	placebo	CBD	placebo	CBD	placebo	CBD	placebo	CBD	placebo	CBD	placebo	CBD
Simple size	12	12	12	12	6	6	6	6	6	6	12	12
Minimum, ng/mL	7.0	6.1	0.1	0.2	2.3	3.7	78.6	140.2	44.0	106.7	0.5	1.5
Maximum, ng/mL	209.6	117.0	1.6	0.7	3.9	5.5	128.6	210.4	116.6	183.0	3.0	3.2
Range, ng/mL	202.6	110.9	1.5	0.5	1.6	1.9	49.9	70.2	72.6	76.2	2.5	1.7
Mean, ng/mL	35.2	24.5	0.9	0.4	3.2	4.6	104.8	163.6	75.6	139.9	1.5	2.2
SD, ng/mL	57.3	30.7	0.4	0.2	0.6	0.8	17.0	26.5	26.6	27.2	0.7	0.7
Mean ratio of CBD/placebo	0.7	​	0.4	​	1.4	​	1.6	​	1.8	​	1.5	​
p value of paired t test	0.2911	​	0.0012	​	0.0079	​	0.0023	​	0.0005	​	0.0001	​

21-OH-Progesterone

A significant decrease in 21-OH-progesterone concentrations was observed in the CBD group compared with the placebo group, with a mean CBD/placebo ratio of 0.44 (Fig. 2; Table 2).

Fig. 2.

Plasma concentrations of 21-OH-progesterone. Left: Plasma 21-OH-progesterone concentrations over time following the final CBD administration. Each point represents the mean of all subjects, and error bars indicate the standard deviation. Dash line: Upper limitation of the physiological range. Right: Paired t test comparing mean 21-OH-progesterone concentrations between the CBD and placebo groups. Each point represents the average hormone concentration.

Testosterone

In all female participants, testosterone levels are all below the lower limit of quantitation. A significant increase in testosterone concentration in male subjects was observed in the CBD group compared with the placebo group, with a mean CBD/placebo ratio of 1.44 (Fig. 3; Table 2).

Fig. 3.

Plasma concentrations of testosterone in male subjects. Left: Plasma testosterone concentrations over time following the final CBD administration. Each point represents the mean of all male subjects, and error bars indicate the standard deviation. Dash lines: Upper and lower limitations of the psychological range. Right: Paired t test comparing mean testosterone concentrations between the CBD and placebo groups. Each point represents the average hormone concentration.

Progesterone

In male subjects, progesterone concentrations were below the assay quantification limit. Due to the fluctuations in progesterone levels across the female menstrual cycle, it was difficult to determine whether CBD administration had any meaningful effect on progesterone concentrations. Only a few female subjects showed a quantifiable plasma concentration for progesterone, and all data are present in Figure 4.

Fig. 4.

Plasma concentrations of progesterone. Each point represents the mean of all subjects, and error bars indicate the standard deviation. Dash line: Upper limitation of the psychological range. Subjects not shown in the graph were below the LLOQ. LLOQ, lower limit of quantification.

Cortisol

Among all subjects, a significant increase in cortisol levels was detected in the CBD group, with a mean CBD/placebo ratio of 1.56 (male) and 1.85 (female) (Fig. 5; Table 2).

Fig. 5.

Plasma concentrations of cortisol. Left: Plasma cortisol concentrations over time following the final CBD administration. Each point represents the mean of all subjects, and error bars indicate the standard deviation. Right: Paired t test comparing mean cortisol concentrations between the CBD and placebo groups. Each point represents the average hormone concentration.

Corticosterone

A significant increase in corticosterone concentrations was observed in the CBD group compared with the placebo group, with a mean CBD/placebo ratio of 1.46 (Fig. 6; Table 2).

Fig. 6.

Plasma concentrations of corticosterone. Left: Plasma corticosterone concentrations over time following the final CBD administration. Each point represents the mean of all subjects, and error bars indicate the standard deviation. Dash lines: Upper and lower limitations of the physiological range. Right: Paired t test comparing mean corticosterone concentrations between the CBD and placebo groups. Each point represents the average hormone concentration.

Discussion

The present study is notable as a novel clinical investigation providing a comprehensive analysis of the effects of CBD on steroid hormones in human subjects receiving MPH. In total, 5 steroid hormones and the steroid precursor cholic acid were quantitatively analyzed in the plasma of 12 participants who received short-term, high-dose oral CBD. For precursor steroids, 21-OH-progesterone concentrations exhibited significant decreases following CBD administration. Among glucocorticoids, cortisol and corticosterone levels were significantly elevated after CBD administration. For reproductive steroids, testosterone concentrations were significantly increased in male subjects following CBD administration (Fig. 7).

Fig. 7.

Summary of the potential effects of CBD on plasma steroid hormones.

Overall, the mechanisms underlying the observed hormonal changes may be related to the role of CB1 receptors in the HPA and HPG axes. CB1 receptors are primarily located on presynaptic GABAergic and glutamatergic neurons within the hypothalamus. When activated by the endocannabinoids anandamide and 2-arachidonoylglycerol, CB1 inhibits voltage-gated Ca²⁺ channels and activates inwardly rectifying K⁺ channels, thereby suppressing neuronal activity [9]. CBD acts as a negative allosteric modulator of CB1; upon binding, it induces structural changes that reduce the CB1 receptor’s binding affinity and signaling efficacy to the endocannabinoids [8]. Consequently, the structural modulation by CBD is thought to increase the activity of GABAergic and glutamatergic neurons, thereby influencing hypothalamic regulation of the HPA and HPG axes (Fig. 7). Beyond the central nervous system, CB1 receptors are also expressed in the adrenal cortex [13] and peripheral neurons [17], indicating their broader role in endocrine regulation. From the result of the current research, the lack of effect of CBD on cholic acid, a precursor not regulated through the hypothalamus, further supports a mechanism mediated primarily via hypothalamic pathways, but not through modifying the cholic acid metabolism.

In the current research, the observed effects of CBD on glucocorticoids included a significant increase in cortisol levels among all subjects. And this elevation agrees with the previous research [18]. Cortisol plays a critical role in the body’s stress response, blood pressure regulation, and immune function [21], and its plasma concentration is widely used as a clinical biomarker of stress [21]. An increasing number of in vitro and clinical exploratory studies have reported anti-stress and anti-inflammatory effects of CBD [22], which are thought to be potentially mediated through CBD’s modulation of the endocannabinoid system within the HPA axis [23]. The present findings provide potential clinical support for the anti-stress pharmacology of CBD. When an experiment testing correlations between CBD exposure (AUC) and hormonal changes [3], cortisol levels exhibited the strongest linear relationship with CBD AUC compared with other hormones (online suppl. Figure 1). Nevertheless, due to the limited sample size, this finding should be interpreted with caution and warrants further investigation.

Further, a significant decrease in 21-OH-progesterone concentrations and a significant increase in corticosterone concentrations were observed following CBD administration. At the tested CBD dose, 21-OH-progesterone concentrations decreased by a factor of 0.44, whereas corticosterone concentrations increased by a factor of 1.46. 21-OH-progesterone, also known as 11-deoxycorticosterone, which is a precursor steroid hormone in the biosynthetic pathway of corticosterone. In humans, its primary biological role is to serve as a precursor of corticosterone in the adrenal cortex [24]. Corticosterone has been shown in vitro to respond predominantly to CB1 receptor activity within the HPA axis, with corticosterone concentrations increasing following selective CB1 receptor antagonism [12, 13]. The current findings may suggest that CBD increases the conversion of 21-OH-progesterone to corticosterone through the neuronal regulation of the HPA axis.

In humans, corticosterone primarily exhibits glucocorticoid activity, with minor mineralocorticoid effects [25]. Elevation in corticosterone concentrations after CBD suggests a potential mechanism through which CBD may reduce stress, possibly by upregulating corticosterone and cortisol levels. On the other side, corticosterone also serves as a key intermediate precursor in the synthesis of aldosterone, the primary bioactive mineralocorticoid [24]. From a clinical perspective, this increase underscores the importance of further investigating CBD’s potential pharmaceutical applications as an anti-stress agent, while also considering possible long-term side effects related to corticosterone’s mineralocorticoid activity during prolonged CBD use.

For reproductive steroids, CBD administration produced an increase in testosterone concentrations in male subjects at the tested dose. Testosterone is a key hormone with essential physiological roles in both reproductive and nonreproductive systems. Circulating testosterone supports the development and maintenance of muscle mass, bone density, physical strength, and reproductive function [26]. Conversely, elevated testosterone levels have also been associated with a higher risk of cardiometabolic disease [27]. Proper testosterone regulation is further important for mental health, mood stability, and cognitive function [28] for men. In this study, male subjects exhibited a marked rise in testosterone following CBD administration. Mean testosterone reached 4.56 ± 0.75 ng/mL (mean ± SD), representing a 1.44-fold increase relative to placebo, while remaining well within the normal physiological range (upper limit: 10.70 ng/mL). Testosterone production is regulated by the HPG axis [14], and this regulation may involve CB1 receptor activity within the hypothalamus. It should be noted that this study did not directly measure upstream regulators of endocrine function, such as adrenocorticotropic hormone, luteinizing hormone, or follicle-stimulating hormone. Consequently, the proposed mechanisms involving the HPA and HPG axes remain speculative.

In addition to modulation through the HPA and HPG axes, several alternative mechanisms may also contribute to the observed changes in hormone concentrations. Beyond CB1 and CB2, additional endocannabinoid receptors have been identified, including transient receptor potential vanilloid 1, although their precise biological functions remain unclear [29]. Furthermore, the hormonal effect of CBD could occur via the peripheral interaction through the adrenal gland. Enzymes such as CYPs and hydroxysteroid dehydrogenases play critical roles in adrenal gland steroidogenesis [30, 31]. While no direct studies have examined CBD’s interactions with specific steroidogenic enzymes, CBD and its metabolites have been shown to inhibit several CYP enzymes, including CYP2C19, CYP2C9, CYP3A, and CYP1A2 [4, 32, 33]. This raises the possibility that CBD may modulate steroid biosynthesis by inhibiting CYP-mediated reactions. CBD could exert its inhibiting effect on stress through modulation of the mesolimbic pathways, where cannabinoids have been reported to exert a significant impact [34, 35], but this possibility was not investigated in the present study. Given the current lack of direct evidence, further research is needed to investigate these potential mechanisms.

The current study has several limitations. First, the sample size was small (n = 12), and the findings require further validation in larger clinical studies. Second, plasma samples were collected only on day 4, which limited the ability to capture CBD’s pharmacodynamic profile and natural daily variations in hormone concentrations. Third, cortisol levels were measured in relative units due to practical constraints, making direct comparisons with published studies challenging. It is important to note that this study incorporated the relatively high oral dose of 1,500 mg of CBD per day in divided doses, an amount that exceeds the doses typically recommended in over-the-counter supplements and many clinical settings. Further studies are therefore warranted to determine whether similar hormonal effects are observed at lower, more commonly used doses of CBD. On the other hand, the widespread use of over-the-counter CBD in various forms and administration routes (e.g., oils, vaping products, and gummies), with unknown bioavailability, suggest the potential for hormonal effects should be considered a possibility from a translational and public health perspective until further assessments can be carried out. Additionally, while 21-OH-progesterone concentrations in human plasma are typically below 0.16 ng/mL [19], the measured concentrations in this study were generally higher, which may be attributable to differences in bioanalytical methods; the present study used LC-MS/MS, whereas previous studies commonly used GC-MS/MS [36]. Finally, several steroid hormones, particularly estrogens, were not assessed due to technical limitations in achieving the required analytical sensitivity.

The original study was designed in part to evaluate potential drug-drug interactions between CBD and MPH [20]. The subject also received 10 mg of MPH, which could, but is unlikely to, alter steroid concentrations. In our protocol, MPH was administered as a single, low dose on the final clinical day in both the CBD and placebo arms, and is unlikely to have produced significant hormonal effects on its own. In healthy adults, acute administration of 60 mg MPH did not affect cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, or testosterone levels over 24 h [37]. However, a potential contribution from the combined exposure to CBD and MPH cannot be excluded and should be acknowledged.

Conclusion

A significant decrease in the precursor steroid 21-OH-progesterone was observed in the CBD group compared with the placebo group. Cortisol and corticosterone levels increased consistently across all CBD-administered participants, and testosterone levels rose significantly in male subjects. No significant differences were identified between the CBD and placebo groups in cholic acid concentrations. All measured hormone levels remained within established physiological ranges. Overall, this study provides novel evidence that CBD modulates key components of the steroidogenic and endocrine systems, highlighting previously unreported endocrine effects of CBD in humans. While there appears to be statistical significance in regard to the level of steroid hormones after the administration of CBD, it remains to be seen whether this will translate to clinical significance. Larger studies should be designed to assess the potential clinical impacts of this CBD dosing regimen.

Statement of Ethics

This study was approved by the Institutional Review Board of University of Florida (trial registration: NCT04603391). All participants provided written informed consent prior to inclusion.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was made possible in part by a grant from the State of Florida Consortium for Medical Marijuana Clinical Outcomes Research.

Author Contributions

Q.Z. statistically analyzed the results and wrote the manuscript. C.A.S. analyzed the plasma samples. P.W.M. statistically analyzed the results. D.J.G., E.N.P., and J.S.M. wrote and reviewed the manuscript.

Funding Statement

This study was made possible in part by a grant from the State of Florida Consortium for Medical Marijuana Clinical Outcomes Research.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.