The Impact of Seasonal Variation on Salivary Hormone Responses During Simulated Mountain Warfare

Abstract

Military personnel routinely complete stressful training exercises in harsh environmental conditions to prepare for intense operational demands. Purpose: This study determined the effect of environmental conditions on salivary hormone profiles in Marines during a mountain warfare training exercise (MTX). Methods: Two cohorts of Marines (age 22 ± 4, height 174 ± 7 cm, body mass 79.2 ± 11.5 kg) completed an MTX (elevation 2100 to 3500 m) in the Fall (n = 63, temperature 11 ± 2 °C) and Winter (n = 64, temperature −5 ± 4 °C). Saliva samples were provided before (PRE), during (MID), and after (POST) the MTX, and were assayed for α-amylase, cortisol, DHEA, testosterone, and osteocalcin. Results: Linear mixed models were used to determine significant interactions (time × season) and found differences in DHEA, testosterone, and osteocalcin. Testosterone and DHEA were lower at MID compared to PRE and POST during the Fall MTX. Testosterone was higher at MID compared to PRE and POST during the Winter MTX, while DHEA remained stable. Osteocalcin was higher in Fall participants compared to Winter but demonstrated a similar trend to increase at MID and decrease at POST in both groups. Cortisol was higher during the Winter MTX compared to the Fall. Conclusions: These findings highlight the differential physiological stress responses in varying seasonal conditions, suggesting the need for tailored training strategies to enhance military readiness and prevent hormonal dysregulation. Further research is needed to elucidate the mechanisms underlying these seasonal effects.

1. Introduction

The Marine Corps is an expeditionary force that rapidly deploys to conduct a broad range of operations while overcoming environmental challenges. To counter the challenges posed by mountainous environments, Marines undergo a specialized Mountain Warfare Training Exercise (MTX) [1]. Over approximately four weeks, Marines learn to overcome obstacles to command and control, firing, maneuverability, and mobility, which are often impaired by the challenging weather and terrain in mountainous regions [2]. These challenges vary considerably with seasons, impacting the operational environment, and requiring Marines to be trained in a range of skills to overcome challenges to even the most basic tactical procedures. For example, traversing rocky terrain, such as scree fields, is extremely difficult as small rocks loosen during ascents in temperate conditions [1]. In contrast, snow can blanket these rugged terrains in the winter, allowing for easier tactical movements if Marines utilize specialized equipment, such as skis and snowshoes [1]. However, the impact of these seasonal variations on the physiological stress experienced by Marines during the MTX has yet to be objectively quantified.

The adaptive physiological response to changing environmental stressors, known as allostasis, is principally governed in the brain by the sympathetic nervous system and the hypothalamic–pituitary–adrenal (HPA) axis, both of which signal endocrine tissues to secrete hormones, peptides, and cytokines to meet the demands of the stressor [3]. These hormone cascades provide insight into the degree of physiological strain encountered during stressful military training, typically resulting in increases in adrenal hormones and reductions in hypothalamic–pituitary–gonadal (HPG) axis hormones [4]. Specifically, military training often results in reductions in testosterone [5,6,7] and increases in cortisol [8,9,10], catecholamines [11,12,13], and DHEA [14,15,16]. Additionally, recent research has established that the skeletal system acts as an endocrine organ, secreting peptides such as osteocalcin, which are instrumental in producing the acute stress response and are sensitive to the demands of military training [16,17,18,19]. Objectively characterizing hormonal changes is crucial, as chronic and extreme military stressors (i.e., allostatic load) can result in several hormonal dysregulation issues including attenuated cortisol awakening responses, hypogonadism, and amenorrhea [20,21,22].

Hormonal dysregulation among military personnel is often linked to prolonged physical exertion coupled with insufficient nutrient intake, and these stressors are thought to be exaggerated by harsh environmental conditions [23]. For instance, a comprehensive review by Tharion et al. concluded that cold weather and high-altitude training environments, similar to those experienced during MTX, are some of the most metabolically taxing conditions faced by military personnel [24]. Therefore, it stands to reason that the combination of cold and mountainous environments may pose an even greater metabolic threat with downstream hormonal consequences. While the metabolic demands of military training in these harsh environments have been the topic of several investigations [25,26,27,28], the neuroendocrine responses have received far less attention. Therefore, the purpose of this study was to determine the effect of environmental conditions on the salivary hormone profiles of Marines during an MTX. We hypothesized that the additional physiological stress from cold exposure encountered during the Winter MTX would increase markers of physiological stress to a greater extent compared to the more temperate conditions of the Fall MTX iteration.

2. Results

Mean ± SE values are provided to depict the between- and within-group differences in the salivary biomarkers for each cohort in Figure 1 and Figure 2, respectively. A significant season-by-time interaction was found for DHEA (F (2, 211) = 10.2, p < 0.0001, η_p² 0.089), which decreased from PRE to MID (p < 0.001) and increased from MID to POST (p < 0.001) in the Fall MTX cohort. DHEA did not significantly change during the Winter MTX. DHEA was higher at MID in the Winter MTX cohort compared to the Fall one (p < 0.001).

A significant season-by-time interaction was found for testosterone (F (2, 211) = 31.6, p < 0.0001, η_p² = 0.23), which increased from PRE to MID (p = 0.01) and decreased from MID to POST (p < 0.0001) during the Winter MTX. Mean testosterone decreased from PRE to MID and increased from PRE to POST during the Fall MTX; while estimated marginal means showed a significant decrease from PRE to MID (p < 0.0001) and a decrease from PRE to POST (p < 0.0001).

No significant interactions were identified for cortisol or α-amylase. Main effects for season were identified for cortisol (F (1, 112) = 6.9, p = 0.01, η_p² = 0.06), which was higher in the Winter MTX compared to Fall at both PRE (p = 0.043) and MID (p = 0.033). Although no main effect of time was identified for α-amylase, an exploratory pairwise comparison of the estimated marginal means revealed a decrease from PRE to POST (p = 0.037). No main effects were found for season on α-amylase.

Time of day when samples were collected did not provide any additional predictive ability to models for osteocalcin, and thus a simpler model was adopted. A significant season-by-time interaction was found for osteocalcin (F (2, 201) = 4.1, p = 0.018, η_p² = 0.04) which was primarily driven by large effects of season (F (1, 114) = 186.2, p < 0.0001, η_p² = 0.62). Both the Fall and Winter MTX had similar effects on osteocalcin. Osteocalcin increased from PRE to MID in Winter (p = 0.044), although this increase was not significant in the Fall MTX. Osteocalcin decreased at POST and was lower than PRE (p = 0.021) and MID (p = 0.012) in the Fall MTX, but these decreases were not significant in the Winter MTX.

3. Discussion

This study assessed the effects of cold exposure on salivary hormone profiles in Marines undergoing a training exercise in a mountainous environment. We hypothesized that the cold exposure during the Winter MTX would induce greater physiological stress compared to the Fall iteration. The major findings indicate differential responses in, suggesting higher stress during the Fall MTX and refute the study hypothesis. However, cortisol levels were higher during the Winter MTX, supporting our hypothesis. Additionally, seasonal differences were observed in osteocalcin, which is implicated in the acute stress response, indicating higher stress during the Fall MTX. Despite these differences, osteocalcin responded similarly in both MTX iterations, warranting further investigations into its role during prolonged stress exposure. Collectively, these data suggest that standardized military training elicits varying physiological stress under different seasonal conditions. Monitoring physiological status through biomarkers, as demonstrated in this study, provides potential “early warning signs” of training maladaptation and may guide targeted interventions [29]. Military leaders could leverage this information to modulate training approaches, prevent hormonal dysregulation, and optimize readiness.

To our knowledge, this is the first study to evaluate the seasonal impact of military training in a mountainous environment using salivary biomarkers of stress. Military training is known to increase adrenal hormones and decrease steroid hormones [4]. We observed reductions in testosterone during the Fall MTX, whereas testosterone increased during the Winter iteration. These differential responses may be partly attributed to seasonal differences in osteocalcin, which increased from PRE to MID during the Winter MTX. Osteocalcin stimulates Leydig cells to secrete testosterone in a bell-curve fashion [30], and participants in the Winter MTX had lower baseline osteocalcin levels, which then increased from PRE to MID. This pattern mirrors the observed increase in testosterone during the Winter MTX, suggesting an osteocalcin-dependent increase in testosterone, consistent with the established bell-shaped response [30].

While direct measures of sympathetic nervous system activity were not assessed, we characterized α-amylase, a non-invasive marker of sympathetic activation. Progressive reductions in α-amylase were observed during both MTX iterations, but only achieved statistical significance in the Winter iteration. These findings differ from our recent reports, which suggest that α-amylase increases in response to simulated military tasks and in response to winter warfare training [22,31,32]. However, it is important to note that α-amylase is rapidly released in response to stress and offers insight into the steady decline observed in the study. The harsh conditions encountered during military training are often associated with severe energy deficits [26,27,33], partly due to voluntary anorexia in cold environments [27]. Such conditions offer potential insights into the observed reductions in α-amylase since athletes with disordered eating secrete less α-amylase daily [34].

DHEA, a testosterone precursor and adrenal hormone, demonstrated a biphasic response in the Fall MTX, unlike the Winter iteration where no changes were observed. DHEA has been extensively studied in military training and is suggested to modulate the negative effects of cortisol [5,9,14]. The DHEA-to-cortisol ratio is often used to assess hormonal imbalance or stress vulnerability [35]. Stress vulnerability may have manifested during the Fall MTX, as DHEA decreased while cortisol remained stable. In contrast, participants in the Winter MTX may have been more stress-resilient, as DHEA remained stable despite elevated cortisol.

This study is the first large-scale investigation of the seasonal impact of standardized military training on salivary hormone profiles. However, there are limitations. Due to resource constraints at the Marine Corps Mountain Training Center and the time-sensitive nature of platoon training objectives, it was not possible for volunteers to participate in both the Fall and Winter MTX iterations. Nonetheless, these conditions reflect real-world training environments. Additionally, the exact details of the training activities during each MTX iteration are not available for publication, and some activities may have been modified due to environmental conditions. While these deviations limit our certainty that the observed differences are due exclusively to the environmental conditions, they represent an ecologically valid characterization of the physiological stress from real-world military training. This study was unable to strictly control the time of day when the saliva samples were collected. However, our statistical analysis accounted for these time of day differences and allowed us to minimize the study’s footprint on the routine military training. Lastly, volunteers were recruited from two separate military installations: Camp Lejeune (Jacksonville, NC, USA), which experiences seasons, and Camp Pendleton (San Diego, CA, USA), which experiences near-constant temperatures. Therefore, we cannot discount the potential effect of cold habituation on the study’s findings.

4. Materials and Methods

4.1. Design

The study protocol was approved by the Naval Health Research Center Institutional Review Board in compliance with regulations to protect human subjects. These data represent a secondary aim of a larger study [36,37] that was approved by the Naval Health Research Center Institutional Review Board (protocol number NHRC.2020.004). This study employed a prospective cohort design in 2 companies of the United States Marines Corps to determine the effect of environmental conditions on salivary hormone profiles during the MTX. Marines attending the Mountain Warfare Training Center’s MTX, between September 2022 and February 2023 were asked to participate in the study. All volunteers were male (n = 126), except for one female, and provided written informed consent prior to the study procedures. All study participants were considered fit for full duty.

The MTX is a routine military training conducted by the United States Marine Corps Mountain Warfare Training Center (Bridgeport, CA, USA). The elevation of the training center’s basecamp is 2100 m with the MTX activities occurring between 2100 and 3500 m. These training activities have previously been described [38]. Briefly, the MTX consists of 3 phases: pre-environmental training, environmental training, and the Marine expeditionary brigade/unit mission rehearsal exercise [1]. Pre-environmental training prepares Marines for environmental considerations (altitude, topography, temperature) and provides them with guidance on specialized clothing and equipment and historical lessons learned. Environmental training emphasizes survival, movement, and fighting skills, in addition to environmental considerations for military tactics, techniques, and procedures. The final MTX phase is a culminating exercise that evaluates dispersed operations in complex, compartmentalized, mountainous terrain that replicates elevations and climates found in the operational environment.

The environmental conditions for the Fall and Winter MTX iterations are provided in

Table 1. Environmental conditions of the Fall and Winter Mountain Training Exercise (MTX) iterations.

MTX Iteration	Average Temperature (°C)	Maximum Temperature (°C)	Minimum Temperature (°C)	Snow Accumulation (m)
Fall	11 ± 2	20 ± 3	3 ± 2	-
Winter	−5 ± 4	7 ± 5	−14 ± 5	20.3

. Data for this study were collected prior to starting the MTX (PRE), at approximately the half-way point of the MTX (MID), and upon completion of the training exercise (POST). MID occurred shortly after the environmental training phase of MTX. Data were collected prior to daily MTX events to control for vigorous physical activity whenever possible. Additionally, subjects abstained from eating, drinking, and using tobacco for 30 min before providing saliva samples.

4.2. Participants

Participants in this study were recruited as part of a larger research project. The volunteers for this study were recruited from Camp Pendleton, CA, and Camp Lejeune, NC, for the Fall and Winter iterations of the study, respectively. Height was measured in centimeters using a stadiometer. Body mass, fat mass, lean body mass, body mass index (BMI), and percent body fat were determined via bioelectrical impedance analysis (InBody 270, Cerritos, CA, USA). Participant demographic information is provided in

Table 2. Participant demographic information for the Fall and Winter Mountain Training Exercise (MTX) iterations.

	Fall MTX (n = 66)	Winter MTX (n = 67)
Age (years)	21 ± 3	23 ± 4
Height (cm)	175 ± 7	174 ± 7
Body Mass (kg)	78.7 ± 10.6	79.7 ± 12.4
Body Mass Index (kg/m2)	25.7 ± 2.7	26.2 ± 3.1
Fat Mass (kg)	13.1 ± 5.1	14.0 ± 5.7
Lean Body Mass (kg)	65.6 ± 8.2	65.7 ± 9.7
Percent Body Fat (%)	16.3 ± 5.1	17.3 ± 5.6

.

4.3. Salivary Hormone Profiles

Saliva samples were collected at PRE, MID, and POST via cotton absorbent oral swab (Salimetrics, Carlsbad, CA, USA). Swabs were placed in the mouth, removed after 2 min, transferred to a conical vial, and placed on ice until transferred to a −20 °C freezer [32]. Salimetrics enzyme-linked immunosorbent assay (ELISA) kits were used to determine the salivary concentrations of dehydroepiandrosterone (DHEA) (sensitivity 5 pg/mL, Item No. 1-1202), total testosterone (sensitivity of 1.0 pg/mL, Item No. 1-2402), cortisol (sensitivity of 0.007 µg/dL, Item No. 1-3002), α-amylase (sensitivity 0.4 U/mL, Item No. 1-1902), and osteocalcin (13.72 pg/mL, Order No. 5212). All samples were assessed in duplicate and reassessed if the between-sample variation exceeded 15%.

4.4. Statistical Analysis

Data were analyzed using R version 4.2.2 (R Core Team 2022). Descriptive statistics (mean ± SE) were reported for all outcome variables. Linear mixed models were used to identify differences in the trajectories of individual markers of physiological stress over time between the Winter versus Fall MTX iterations. Models included the subject as a random effect, and season, time, and the season-by-time interaction as fixed factors. Time of day was also evaluated as a fixed factor in all metrics to control for deviations in the time when samples were collected, which were outside of the researchers’ control. Variables were screened for potential outliers and normality violations prior to analysis and log-transformed when normality violations occurred. Model fit was evaluated using Akaike information criterion (AIC). Nakagawa & Schielzeth R² and omnibus F tests were reported for the variability explained by each model. Benjamani–Hochberg corrections were employed in pairwise post hoc tests to limit the false discovery rate in the presence of any significant main effects or interactions. Statistical significance was set at α = 0.05.

5. Conclusions

Novel insights into the physiological stress responses of Marines undergoing mountain warfare training in different seasonal conditions were explored in this study. Our findings demonstrate that standardized military training elicits varying hormonal responses depending on the environmental context, with greater stress (DHEA, testosterone) observed during the Fall MTX, but elevated cortisol during the Winter MTX. The differential testosterone and DHEA responses, as well as the role of osteocalcin, highlight the complex interplay between environmental stressors and neuroendocrine status. These findings underscore the importance of physiological monitoring to identify potential hormonal dysregulation and optimize military training. Future research should further explore the mechanisms behind these seasonal variations, contributing to a better understanding of how environmental factors influence training stress and potentially offering valuable insights for enhancing military readiness and performance.