*2.8. Saliva Samples*

The PD method requires participants to sit with his or her head flexed forward while saliva passively drips into a container [18]. Participants stored the samples in their refrigerator (4 ◦C) until day 2, where they brought the samples to the rock-climbing gym. Approximately 3 mL of saliva was collected.

Salivary cortisol samples were stored at −80 ◦C until analysis. They were then centrifuged at 2500× *g* for 10 min, and 1.5 mL of the separated samples was placed in Eppendorf microtubes (Starsledt Akhengesellshaft & Co., Nümbrecht, Germany). Salivary cortisol was measured with a Cortisol Saliva Enzyme-Linked Immunosorbent Assay (ELISA) procedure (IBL International GMBH, Hamburg, Germany).

#### *2.9. Statistics*

All analyses were performed using the statistical package IBM® SPSS® Statistics Software (SPSS) version 26. Results are expressed as mean ± standard deviation. After verifying that all values were within the normal range, *T* tests were performed to compare the mean values of different conditions. A one-way ANOVA analysis was used to compare the variables measured between sexes and between test conditions. Pearson's correlation was utilized to analyze the relationship between time to fatigue and body weight, as well as the number of pull-ups and cortisol levels. Differences were considered significant at *p* < 0.05.

#### **3. Results**

#### *3.1. Cortisol*

No outliers in cortisol concentrations were identified; however, values that were not within the reportable range of 0.015–3.00 μg/dL were discarded, as indicated by the Cortisol Saliva ELISA kit (IBL International GMBH, Germany). Baseline cortisol concentrations were highest at 8:00 a.m., with average values of 0.71 μg/dL ± 0.35. There were significant differences between cortisol concentrations at 8 a.m. for the three baseline measurements in males (0.78 μg/dL ± 0.47, *p* = 0.00; 0.59 μg/dL ± 0.43, *p* = 0.002; 0.80 μg/dL ± 0.56, *p* = 0.003), as well as when compared to pre-startle (1.85 μg/dL ± 0.70, *p* = 0.000), poststartle (1.73 μg/dL ± 0.67, *p* = 0.000), and post-fatigue (1.44 μg/dL ± 0.61, *p* = 0.000). There were no significant differences in cortisol concentrations in males when comparing the three test conditions (1.85 μg/dL ± 0.70, 1.73 μg/dL ± 0.67, 1.44 μg/dL ± 0.61).

Post-startle cortisol concentrations were significant (1.57 μg/dL ± 0.96, *p* = 0.046) for females when compared with pre-test levels (1.26 μg/dL ± 0.29), and there were also significant differences (*p* = 0.043) between female pre-startle (1.26 μg/dL ± 0.29) and post-fatigue (1.37 μg/dL ± 1.12). There were significant differences (*p* = 0.050) in males and females between pre-startle (1.72 μg/dL ± 0.66) and post-fatigue (1.42 μg/dL ± 0.72) cortisol levels. Cortisol baseline concentrations are shown in Figure 1, and concentrations in the different test conditions are shown in Figure 2.

**Figure 1.** Mean baseline salivary cortisol concentrations expressed in μg/dL ± SD from the three days of sampling at 8:00 a.m., 11:00 a.m., and 2:00 p.m. (**a**) Results for males and females. (**b**) Results for males. (**c**) Results for females.

There were no significant differences between sexes in cortisol concentrations in the pre-startle (1.85 μg/dL ± 0.70, 1.26 μg/dL ± 0.29), post-startle (1.73 μg/dL ± 0.67, 1.57 μg/dL ± 0.96), and post-fatigue (1.44 μg/dL ± 0.61, 1.37 μg/dL ± 1.12) conditions. Based on the ANOVA F analysis, there was a positive correlation between number of pull-ups and pre-test cortisol concentrations (*p* = 0.008, r = 0.814, R2 = 0.663, CI = 95%).

**Figure 2.** Mean cortisol concentrations expressed in μg/dL ± SD from the three test conditions of day 2. (**a**) Results for males and females. (**b**) Results for males. (**c**) Results for females.
