*3.3. FSST*

There were no significant differences between FSST values baseline pre-climb day 1 (3.62 s ± 0.75) and post-startle day 2 (3.09 ± 0.62). There were significant differences (*p* = 0.006) between FSST scores baseline post-climb day 1 (3.45 s ± 0.61) and post-startle day 2 (3.09 s ± 0.62), and significant differences (*p* = 0.002) between males' baseline postclimb day 1 (3.42 s ± 0.53) and post-fatigue day 2 (2.84 s ± 0.43) scores. Results are shown in Tables 3–5.

**Table 3.** Summary of results for males and females. Significant differences (*p* = 0.003) in somatic anxiety post-startle between males and females. Significant differences (*p* = 0.019) between male and female self-confidence values pre-startle and post-fatigue (*p* = 0.02). Significant differences (*p* = 0.006) in FSST times between baseline post-climb and post-startle. Significant differences (*p* = 0.012) between baseline pre-climb left-hand grip strength and post-fatigue left-hand grip strength.


**Table 4.** Summary results for males. Significant differences (*p* = 0.019) in somatic anxiety pre- and post-startle in males. Significant differences (*p* = 0.002) between FSST baseline post-climb and post-fatigue scores. Significant differences (*p* = 0.035) between baseline pre-climb left-hand grip strength and post-fatigue left-hand grip strength. Significant differences (*p* = 0.00) between pre-startle and post-startle right-hand grip strength.


**Table 5.** Summary results for females.


#### *3.4. Anxiety and Self-Confidence*

There were significant differences (*p* = 0.019) in somatic anxiety pre-startle (14.23 ± 5.09) and post-startle (16.36 ± 5.54) in males, as well as significant differences (*p* = 0.035) between male and female self-confidence levels pre-startle (35.08 ± 4.94, 30.00 ± 5.29). There were also significant differences (*p* = 0.022) in self-confidence post-fatigue between sexes (34.43 ± 5.88, 29.00 ± 4.76). Results are shown in Tables 3–5.

#### *3.5. Grip Strength*

There were significant differences (*p* = 0.012) between baseline left-hand grip strength (48.60 kg ± 9.36) and post-fatigue left-hand grip strength (36.46 kg ±8.63). Males had significant differences (*p* = 0.035) between baseline left-hand grip strength (52.27 kg ± 6.94) and post-fatigue left-hand grip strength (35.35 ± 14.74). There were differences in female baseline left-hand grip strength (38.50 kg ± 7.94) and post-fatigue left-hand grip strength (29.25 kg ± 2.36), although not significant (*p =* 0.058). Males also had significant (*p =* 0.00) differences in pre-startle (50.55 ± 10.48) and post-startle (51.05 ± 8.11) right–hand grip strength. Results are reported in Tables 3–5.

#### *3.6. Fatigue*

There was an inverse correlation between time to fatigue and body weight (CI = 95%, r = 0.606, *p =* 0.025). There were significant differences (*p =* 0.022) between sexes: males reached muscular failure after 282.39 s ± 48.20, and females after 367.51 s ± 70.21.

#### **4. Discussion**

The results indicate that physical and psychological stress affects males and females in different ways and that cortisol concentrations are strongly affected by time of day. Salivary samples were utilized in this study because since cortisol follows a circadian rhythm, with the highest values occurring 20–40 min after waking, we thought it beneficial to obtain a baseline secretion curve for comparison with the rest of the values [18,19]. Baseline cortisol concentrations followed a normal diurnal pattern, with the highest values occurring at 8:00 a.m.

It is possible that the variations in concentrations between the 8:00 a.m. samples of each day were due to individual error or individual variation in diurnal cortisol slope (DCS). Cortisol concentrations can be easily affected by acute stressors, age, sex, nutrition, sleep, hydration, physical activity, and circadian rhythm [4,9,18]. Salivary composition can also be affected by countless factors, including circadian rhythm, age, sex, smoking, diet, and medications [4]. Since external factors of the participants' day to day were not accounted for, it is possible that variations in these variables altered their DCS. In addition, it is possible that participants did not take the samples at the same time for each of the three baseline days. Although these may have been small variations, it may have been enough to affect the DCS substantially—especially in the waking hours [19,20]. Variations in sampling time may have also been due to difficulty in saliva production. Some participants reported spending 20 min in the PD position to produce sufficient saliva. This may have further delayed the time of day that the sample was obtained, thus influencing cortisol levels. This may have also been a factor on day 2, since some participants took substantially longer to produce enough saliva pre-startle, post-startle, and post-fatigue. Although the pre-startle samples were taken at 8:00 a.m., and the post-startle and post-fatigue samples were taken shortly after, time of day may have profoundly impacted the variance in salivary cortisol levels. Since the fatigue climb was the last test condition, this could explain the decrease in cortisol levels in males and females. Instead of obtaining baseline samples at 8:00 a.m., 11:00 a.m., and 2:00 p.m., perhaps obtaining samples at 8:00 a.m. and 9:00 a.m. would have been a better comparison for this study. Moreover, lead climbing and the auditory stimulus used may not have been strong enough stressors to provoke changes in cortisol levels in males due to their level of experience and more advanced skill level [1].

The 8:00 a.m. pre-startle cortisol levels were higher in both males and females when compared to their respective baseline 8:00 a.m. cortisol levels. This may have been due to an anticipatory cortisol response that primes the central nervous system [21]. This anticipatory response provides some insight into the relationship between psychological stress and physiological responses, as well as highlights the significance of psychobiological processes that occur prior to a stressor. It is possible that this neuroendocrine response was activated when instructions for the startle climb were provided. This would suggest that the stress (and increase in cortisol) that individuals experienced was triggered by their emotional and cognitive representations of what they thought would occur during the climb [21].

Females may have experienced a peak in cortisol levels post-startle because the relative difficulty of the climb may have been higher for them. Female participants were not as comfortable with the lead climbing technique, and this lack of confidence, in addition to the sustained isometric contractions and increasing difficulty of the climb, may have contributed to a peak in cortisol levels post-startle. This is supported by the significant differences in self-confidence between males and females prior to the startle climb.

Increased somatic anxiety post-startle in males may have been due to the added stress of lead climbing. Other studies have had similar findings, noting that participants had increased somatic anxiety when they had to lead climb a route, compared to top rope climbing [2]. Sex differences in somatic anxiety post-startle may be an indicator of differences in male and female responses to stress. There is evidence from functional magnetic resonance imaging (fMRI) that women are more attuned to negative stimuli and that they respond more rapidly to negative stimuli [22]. These sex differences may also explain differences in the self-reported self-confidence post-fatigue climb.

FSST times may have been faster post-startle because of heightened somatic anxiety and focus, due to the fight-or-flight response. It is also possible that the results were influenced by test familiarization and decreased anxiety of social judgment. In day 1, the FSST trials were carried out when the rock-climbing gym was open to the public. Therefore, there were other climbers present that served as an "audience" to the participants in the study. The participants may have also had difficulty focusing on the task at hand because of the various distractions in the gym. The fact that the participants did not know what to expect, that it was their first time performing the FSST, that there was an audience, and that their focus could have been affected, may have all contributed to slower day 1 scores.

It could be that average FSST times did not decrease post-fatigue because the value that was used to indicate fatigue was forearm muscle failure. It may be that although the forearm musculature fatigued to failure, focus and lower limb coordination did not decline. It is also worth noting that three participants were not able to complete the test post-fatigue, due to poor coordination and unsuccessful execution of the sequence.

#### **5. Conclusions**

Our results show that cortisol concentrations follow a normal standard curve, irrespective of the test condition. Cortisol samples were taken 15 min after the stressor, and values were lower post-startle and post-fatigue when compared to the pre-test. It may be that the stressors used in this study were not enough to provoke a stressful situation in the climbers of this study or that higher values were presented immediately after the climb and not 15 min later. Future studies should compare the cortisol response immediately after the stimulus, as well as 15 min later, to determine when the true peak in cortisol occurs. Studies should also look at ways to reduce the amount of time spent in saliva sampling, since extended sampling time may have profoundly affected cortisol levels.

There seem to be differences in the way that males and females psychologically prepare and react to stressful situations. It is difficult to draw conclusions from this sample because a major limitation was the number of participants, especially females. Evidently, there are countless factors that can influence the stress response during climbing, as well as several variables that can serve as indicators of the demand of the climb. Future studies should also take into consideration the biomechanical and strategic changes that occur with increased psychological and physiological stress. This can be done by analyzing video footage and utilizing electromyography (EMG) to determine premotor time and reaction time, as well as changes in muscle activity. Blood samples can also be taken to look at the impact that acute stressors have on biomarkers of oxidative stress, as well as on biomarkers that are suggested to be related to anxiety [4].

**Author Contributions:** Conceptualization, P.V. and C.B.; methodology, P.V., A.I. and C.B.; formal analysis, C.B.; investigation, P.V. and C.B.; resources, C.B.; writing—original draft preparation, P.V.; writing—review and editing, P.V., C.B. and S.S.; visualization, P.V., C.B. and S.S.; supervision, C.B. and A.I.; project administration, C.B. and A.I.; funding acquisition, C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to the observational nature of the study.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Carles Ros and Carles Tudela and the team at Ingravita for their support and collaboration on the project. The authors would like to extend their gratitude to Albert Pare for his continued assistance and valuable insights during the investigation, to Sergio Vilches for his contribution in the development of the project and for providing resources for saliva analysis, to Jon González for his help with sample analysis, and to all the climbers that participated in the study, for their cooperation and commitment to the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

