*3.1. Acclimation*

We did not find changes across sex (*p* = 0.16) or time (*p* = 0.10) with the corticosterone EIA (Figure 2A). Similarly, there were no changes across sex (*p* = 0.60) or time (*p* = 0.22) with the group-specific EIA (Figure 2B). However, it is noteworthy that the variability in FCMs appeared smaller towards the end of the dark cycle compared to the beginning. FCMs collected during the third day of acclimation were considered as baseline when we evaluated the effects of treatments (dexamethasone and ACTH).

**Figure 2.** Changes in corticosterone baseline fecal cortisol/corticosterone metabolites (FCMs) from laboratory-bred deermice across the dark cycle measured with (**A**) corticosterone enzyme immunoassay (EIA) and (**B**) group-specific EIA. Lines connect means from each sampling time and circles indicate data points whereby an individual is denoted with a different color. Males are shown in blue and green colors, whereas females are shown in red and tan.

#### *3.2. Dexamethasone Suppression Test*

We found a sampling time by treatment effect for the corticosterone EIA (*F*4, 18.31 = 3.73, *p* = 0.02; Figure 3A). Deermice had lower FCM levels (*n* = 4, 6.23 ± 0.26 ln ng/g) ~10 h post injection than baseline (06:00 h, *n* = 3, 7.54 ± 0.30 ln ng/g, post hoc, *t*18.32 = −3.37, *p* = 0.003). We also found a sampling time by treatment effect for the group-specific EIA (*F*4, 21 = 3.39, *p* = 0.03; Figure 3B). Deermice had lower FCMs (*n* = 4, 7.03 ± 0.15 ln ng/g) than baseline ~10 h post injection (06:00 h, *n* = 3, 7.60 ± 0.17 ln ng/g, post hoc, *t*19.46 = −2.47, *p* = 0.02). Interestingly, deermice showed a marginal increase in FCMs (*n*

= 4, 8.16 ± 0.15 ln ng/g) ~4 h post injection compared to baseline (*n* = 2, 7.63 ± 0.22 ln ng/g, *t*20.42 = 1.96, *p* = 0.06). We note that baseline FCM levels at 06:00 h represent a 2 h interval (04:00–06:00 h) whereas treatment FCM levels at the same time representa4h interval (02:00–06:00 h; Table 1).

**Figure 3.** Corticosterone baseline fecal cortisol/corticosterone metabolites (FCMs) (solid lines and circles) versus post dexamethasone FCMs (dashed lines and triangles) in laboratory-bred deermice measured with (**A**) corticosterone enzyme immunoassay (EIA) and (**B**) group-specific EIA. Lines connect means from each sampling time. Circles and triangles indicate individual data points whereby an individual is denoted with a different color. Males are shown in blue and green colors, whereas females are shown in red and tan. Dexamethasone was administered at ~20:00 h (not shown). Asterisk (\*) denotes significant differences at α = 0.05.

#### *3.3. ACTH Stimulation Test*

We found an effect of treatment for the corticosterone EIA (*F*1, 19.22 = 11.94, *p* = 0.003; Figure 4A) where deermice had consistently higher FCM levels (7.77 ± 0.19 ln ng/g) post injection than baseline (7.27 ± 0.20 ln ng/g, post hoc, *t*19.17 = 3.44, *p* = 0.003). We also found an effect of sampling time (*F*4, 19.19 = 5.42, *p* = 0.004) where deermice had higher FCM levels at 22:00 h (7.84 ± 0.23 ln ng/g; *t*19.07 = 4.06, *p* = 0.005), 00:00 h (7.74 ± 0.24 ln ng/g; *t*19.17 = 3.45, *p* = 0.02), and 06:00 h (7.62 ± 0.22 ln ng/g; *t*19.05 = 3.14, *p* = 0.04) compared to 08:00 h (6.99 ± 0.22 ln ng/g) regardless of treatment. Similarly, using the group-specific EIA, we found an effect of treatment (*F*1, 20.25 = 14.16, *p* = 0.001; Figure 4B) where deermice had higher (7.99 ± 0.12 ln ng/g) FCM levels consistently post injection than baseline (7.58 ± 0.12 ln ng/g, *t*20.16 = 3.75, *p* = 0.001; Figure 4B). Again, we also found an effect of sampling time (*F*4, 20.28 = 3.42, *p* = 0.03) where deermice had higher FCM levels at 22:00 h (8.02 ± 0.15 ln ng/g) compared to

08:00 h (7.48 ± 0.14 ln ng/g, *t*20.06 = 3.50, *p* = 0.02) regardless of treatment. We note that baseline FCM levels at 06:00 h representa2h interval (04:00–06:00 h) whereas treatment FCM levels at the same time representa4h interval (02:00–06:00 h; Table 1).

**Figure 4.** Corticosterone baseline fecal cortisol/corticosterone metabolites (FCMs) (solid lines and circles) versus post adrenocorticotropic hormone (ACTH) FCMs (dashed lines and triangles) in laboratory-bred deermice measured with (**A**) corticosterone enzyme immunoassay (EIA) and (**B**) group-specific EIA. Lines connect means from each sampling time. Circles and triangles indicate individual data points whereby an individual is denoted with a different color. Males are shown in blue and green colors, whereas females are shown in red and tan. ACTH was administered at ~20:00 h (not shown).

## *3.4. Field Validation*

For field study 1 (group-specific EIA), we found that there was a marginal effect of confinement time on FCM levels (*F*2,15 = 3.38, *p* = 0.06), which we still elected to explore because of likely biological relevance. We found no effect of sex (*p* = 0.56) or reproductive status (*p* = 0.39). Deermice confined for 4–6 h had marginally higher FCM levels (*n* = 6, 4 females and 2 males, 8.33 ± 0.08 ln ng/g) compared to those confined for 0–2 h (*n* = 7, 4 females and 3 males, 7.90 ± 0.11 ln ng/g, *p* = 0.07). However, deermice confined for 0–2 h had no differences in their FCM levels compared to those of deermice confined for 8–10 h (*n* = 7, 5 females and 2 males, *p* = 0.16) or 4–6 h versus 8–10 h (*p* = 0.85; Figure 5). For field study 2 (corticosterone EIA), we found a significant effect of confinement time on FCM levels (*F*1, 10 = 23.21, *p* = 0.001). Deermice had lower FCMs (9.12 ± 0.32 ln ng/g) when confined for 0–4 h compared to after short-term restraint and overnight confinement (*n* = 7, 11.10 ± 0.32 ln ng/g, *t*6 = 4.82, *p* = 0.003; Table 2). Sex (*p* = 0.60) and reproductive status (*p* = 0.75) were not significant.

**Figure 5.** Fecal cortisol/corticosterone metabolites (FCMs) across different trap confinement times in free-ranging deermice. FCMs were measured with the group-specific EIA. Boxplots display the median (line), 25–75% interquartile range (boxes) and the full range (whiskers). In the 0–2 h group, there is a large outlier. Circles indicate individual data points whereby blue denotes males and red denotes females.

**Table 2.** Summary of demographic factors and fecal cortisol/corticosterone metabolite (FCM) values from free-ranging deermice captured in MT, USA in August 2017 for field study 2.


1 Reproductive status was determined via scrotal testes in males and presence of a perforate vagina, lactation, or pregnancy in females, 2 FCM difference was calculated by subtracting FCMs at 0–4 h from FCMs overnight.
