High-altitude mountaineering represents an extreme physical and psychological challenge that combines multiple stressors, including hypoxia, cold exposure, physical exhaustion, sleep fragmentation and psychological pressure [1,2,3]. While many individuals successfully complete such expeditions, others experience significant psychological distress, raising questions about individual susceptibility and the biological mechanisms underlying stress responses in extreme environments. Understanding these individual differences is critical not only for optimizing climber safety and performance but also for identifying those who may require additional support or modified protocols. Several recent overviews summarize molecular, neurophysiological and clinical evidence suggesting that acute and chronic altitude exposure can alter neurotransmitter systems, cerebral perfusion and neural network function, with downstream effects on cognition and affect [4]. Recent evidence underscores the psychological burden associated with high-altitude exposure. Large-scale prospective data from the Intern Health Study demonstrated that individuals living at higher elevations reported a significantly increased risk of depression and anxiety compared to those at sea level, suggesting a dose–response relationship between altitude and mood disturbance [5]. In line with these findings, a recent systematic review and meta-analysis reported that the prevalence of depressive symptoms is substantially higher in high-altitude populations, with heterogeneity across studies but a consistent overall signal of increased vulnerability [6]. The role of acute mountain sickness (AMS) in mediating these affective outcomes has also been highlighted. A study identified anxiety, irritability and depression as both predictors and correlates of AMS severity, suggesting a bidirectional relationship between somatic symptoms of altitude illness and psychological distress [7]. Nevertheless, despite accumulating epidemiological and clinical evidence, targeted field studies using standardized psychometric tools remain relatively scarce, and the neurobiological mechanisms underlying these changes are only beginning to be explored [7].

The endocannabinoid system (ECS) plays a crucial role in stress regulation, mood modulation, and adaptation to environmental challenges, such as hypoxia and high-altitude exposure [8,9]. Two key components of this system are the cannabinoid receptor type 1 (CNR1/CB1) and fatty acid amide hydrolase (FAAH), the primary enzyme responsible for degrading anandamide, the brain’s endogenous cannabinoid. Variations in the activity of these genes have been associated with individual differences in stress resilience, anxiety responses, and psychological well-being [10,11,12]. Importantly, the ECS exhibits marked sexual dimorphism in both baseline function [13] and stress responsivity, with males and females showing distinct patterns of endocannabinoid signaling and receptor expression under stress conditions [14,15]. Females are more sensitive than males to the effects of cannabinoids, and many sexually dimorphic effects can be attributed to a sexually dimorphic ECS [13]. In addition, the ECS contributes to cerebrovascular regulation. Endocannabinoids modulate vascular tone and cerebral blood flow, processes that are particularly relevant under hypoxic stress, when oxygen delivery to the brain is compromised. Craft et al. (2012) reported that ECS activity influences neurovascular coupling, suggesting that alterations in this pathway could contribute to cognitive and affective changes associated with high-altitude exposure [13]. van Doorslaer de Ten Ryen et al. (2023) showed that both endurance and resistance exercise in hypoxia alter the levels of endocannabinoids and the expression of ECS-related enzymes, highlighting a role for ECS signaling in integrating metabolic and vascular responses during physical exertion in oxygen-limited environments [16]. Males have higher CB1 receptor binding sites than females, but females possess more efficient CB1 receptors, and this consistent dimorphism in the ECS may justify the different sensitivity observed between sexes in behavioral paradigms concerning emotion and cognition [17]. Moreover, chronic unpredictable stress exposure results in sexually dimorphic changes in CB1 receptor density, with male rats showing decreased hippocampal CB1 receptor density while female rats exhibit a significant increase following the same stress exposure [18]. Sex differences exist with regional selectivity in the neuroanatomical arrangement of the ECS, and endocannabinoids exhibit sexual dimorphism in serum concentrations across the lifespan [19]. These sex differences may contribute to the differential prevalence and presentation of stress-related psychiatric disorders observed between men and women.

Beyond its classical biochemical regulation, the ECS is also influenced by epigenetic mechanisms, particularly DNA methylation and microRNA (miRNA) regulation, which provide dynamic control over gene expression without altering the underlying DNA sequence [20]. These mechanisms are increasingly recognized as critical mediators of stress adaptation, capable of responding to environmental challenges and potentially predicting individual vulnerability to stress-related disorders [21]. DNA methylation, the addition of methyl groups to cytosine residues of CpG dinucleotides in CpG islands, is a fundamental mechanism in transcriptional repression [22]. MicroRNAs provide a complementary post-transcriptional regulatory mechanism of gene expression: these small non-coding RNAs bind to specific messenger RNAs (mRNAs) to inhibit translation or promote degradation [20]. The epigenetic landscape itself shows sex-specific patterns with males and females often displaying divergent epigenetic responses to the same environmental stressors [23]. Previous research has identified specific miRNAs, including miR-23b-3p (which directly targets CNR1) and miR-4505 (associated with psychiatric outcomes), as potential biomarkers in stress-related contexts [24,25,26]. Moreover, coordinated regulation through multiple epigenetic mechanisms—such as simultaneous changes in DNA methylation and miRNA expression [27]—may provide more robust insights into stress adaptation than single-marker approaches.

The present pilot and explorative study investigated epigenetic changes in the FAAH and CNR1 genes in Italian mountaineers participating in a Himalayan expedition. Saliva samples were collected before ascent (T0) and after ascent (T1) from both male and female participants, allowing for sex-stratified analysis of epigenetic responses. Given that several subjects reported psychological stress at T1, and considering the established sexual dimorphism in both stress responses and ECS function, we hypothesized that: (1) high-altitude exposure would induce measurable epigenetic changes in FAAH and CNR1 genes; (2) these changes would differ between males and females, reflecting sex-specific patterns; (3) epigenetic changes might vary between individuals who maintain psychological resilience and those who experience distress; and (4) directional concordance between DNA methylation and miRNA expression patterns might suggest CNR1 as a candidate regulatory target in high-altitude-related stress responses.

These preliminary findings may contribute to informing future sex-specific research designs and help generate hypotheses regarding potential epigenetic indicators of psychological vulnerability to extreme altitude activities, pending validation in larger and adequately powered cohorts.