Hair cortisol detection in dairy cattle by using EIA: protocol validation and correlation with faecal cortisol metabolites

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein–Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R²=0.98 and mean percentage error of −10.8±1.55%. The extraction efficiency was 89.0±23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.     

Relocation and Hair Cortisol Concentrations in New Zealand White Rabbits

To investigate how long relocation modified hair cortisol concentrations in New Zealand white rabbits, 19 rabbits were subjected to a change in their breeding facility at the beginning of the trial and then were kept under stable environmental conditions. Hair samples were collected at the time of arrival to the nonhuman animal facility and at 40-day intervals from the same skin area for up to 440 days after the animals' arrival to the facility. A period effect on the hair cortisol concentration was found (p < .01). The transfer of the rabbits to the new facility might have induced an increase in the hypothalamic-pituitary-adrenal axis activity (p < .01). A second increase in hair cortisol concentration (p < .01) occurred at 320 days, after a change of personnel at the facility that occurred at 280 days, which was the only environmental change. The relocation of rabbits to the facility resulted in a stress response leading to elevated cortisol levels. The effect of relocation on mean cortisol concentrations was exhausted within 120 days when all environmental factors were kept stable.     

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT "point samples" obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair''s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.     

Bobcat Hair Cortisol Correlates with Land Use and Climate

Bobcats (Lynx rufus) have been increasing in abundance in the northeast United States despite a corresponding trend of increased anthropogenic land uses. Inhabiting areas of high human land use can affect stress levels, and hence cortisol titers, for wildlife species by increasing frequency of human interaction and altering habitats. In turn, increased cortisol levels can have negative effects at the individual and population level including decreased immune function, slowed growth and tissue repair, reduced reproductive capacity, and nutritional deficiencies. We quantified cortisol in bobcats across New Hampshire and Vermont, USA, using hair samples, then explored associations between hair cortisol and various organismal, land use, land cover, and climatic variables at 2 different spatial scales. Hair cortisol differed by season and bobcat mass. On average, cortisol levels were higher in fall than in spring, and larger bobcats had lower cortisol levels. Anthropogenic land uses—especially residential and agricultural uses—were the most important predictors of hair cortisol at the town scale ( area = 93 km²). At a larger scale (Wildlife Management Units; area = 1,256 km²), temperature and precipitation were better predictors of hair cortisol, suggesting that extreme weather may have significant effects on bobcat population dynamics. Our results highlight the importance of landscape composition and local conditions in the sustainable management of furbearer populations. © 2021 The Wildlife Society.     

Effect of environmental enrichment and herbal compound supplementation on physiological stress indicators (chromogranin A,cortisol and tumour necrosis factor-α) in growing pigs

Stress response induces physiological, behavioural, immunological and biochemical changes that directly affect health and well-being. Provision of environmental enrichment and herbal compounds may reduce stress in current commercial pig husbandry systems. The aim of this study was to evaluate the effect of providing different environmental enrichment materials (EE) and a herbal compound (HC) on physiological indicators of acute and chronic stress in growing pigs (salivary cortisol and chromogranin A (CgA), hair cortisol and tumour necrosis factor-α (TNF-α)). Salivary cortisol and CgA have been reported as biomarkers basically of acute stress, whereas hair cortisol and TNF-α have been more related to chronic stress. For this purpose, eight groups of seven pigs each (14 pigs/treatment, 56 pigs in total) were used: (a) two EE groups, (b) two groups supplemented with HC, (c) two groups provided both with EE and HC and (d) two control groups. Samples of hair, saliva and blood were taken to measure cortisol (in hair and saliva), CgA (in saliva) and TNF-α (in blood) at three different times: before starting the experiment (T0), and after 1 (T1) or 2 months (T2) of providing the materials and herbal compound. No differences were found at T0 in salivary or hair cortisol, CgA or TNF-α, whilst at T2, the control group showed significant increased concentrations of CgA and hair cortisol, when compared with the rest of the treatments (P<0.001). These differences were significant at T1 only for CgA (P<0.001). Furthermore, an overall correlation was reported between hair cortisol and salivary CgA (r=0.48, P<0.001). These results support that providing enrichment material or an herbal compound may reduce stress in growing pigs. Furthermore, the results support that hair cortisol and CgA may be proper non-invasive tools to detect stress, specially associated with factors of chronic exposure.     

Quantification of hair cortisol concentration in common marmosets (Callithrix jacchus) and tufted capuchins (Cebus apella)

Quantifying cortisol concentration in hair is a non‐invasive biomarker of long‐term hypothalamic‐pituitary‐adrenal (HPA) activation, and thus can provide important information on laboratory animal health. Marmosets (Callithrix jacchus) and capuchins (Cebus apella) are New World primates increasingly used in biomedical and neuroscience research, yet published hair cortisol concentrations for these species are limited. Review of the existing published hair cortisol values from marmosets reveals highly discrepant values and the use of variable techniques for hair collection, processing, and cortisol extraction. In this investigation we utilized a well‐established, standardized protocol to extract and quantify cortisol from marmoset (n = 12) and capuchin (n = 4) hair. Shaved hair samples were collected from the upper thigh during scheduled exams and analyzed via methanol extraction and enzyme immunoassay. In marmosets, hair cortisol concentration ranged from 2,710 to 6,267 pg/mg and averaged 4,070 ± 304 pg/mg. In capuchins, hair cortisol concentration ranged from 621 to 2,089 pg/mg and averaged 1,092 ± 338 pg/mg. Hair cortisol concentration was significantly different between marmosets and capuchins, with marmosets having higher concentrations than capuchins. The incorporation of hair cortisol analysis into research protocols provides a non‐invasive measure of HPA axis activity over time, which offers insight into animal health. Utilization of standard protocols across laboratories is essential to obtaining valid measurements and allowing for valuable future cross‐species comparisons.     

The age of hair matters – the incorporation of cortisol by external contamination is enhanced in distal hair segments of pigs and cattle

Hair cortisol concentration (HCC) is used as an indicator of long-term stress or pathologies in humans and increasingly in animals. Although the main mechanism for the incorporation of cortisol into the hair shaft is by diffusion from blood, cortisol may also be incorporated from external sources by contamination of the hair surface. In farm animals under conventional husbandry conditions and trapped animals, contamination of hair with cortisol-containing body fluids, especially with urine, was shown to be a considerable confounding factor when studying HCCs. We recently found that cattle and pigs exhibit elevated HCCs in distal hair segments and assume that the incorporation of external cortisol is facilitated in these older hair segments. Therefore, the aim of this study was to investigate the effects of urine contamination on HCC in different hair segments of pigs and cattle, and to determine whether different cleaning protocols can prevent contamination effects. In an in vivo experiment in pigs (n = 18) and an in vitro experiment in cattle (n = 12), hairs were repeatedly contaminated with urine of the respective species and then shaved or cut in segments. Cortisol concentrations in hair segments were analysed by enzyme immunoassay after washing with isopropanol and extraction with methanol. Results were compared with HCCs in untreated hairs or hairs treated with water. Moreover, additional bovine hair samples contaminated with urine were subjected to two further cleaning procedures. Contamination with urine generally increased HCCs, and it was demonstrated for the first time that this effect is more pronounced in distal compared to proximal hair segments in both species. The immersion of bovine hair in vitro in water caused a washout of cortisol, which was also more pronounced in distal hair segments. In general, the different cleaning protocols for cattle hair did not prevent contamination effects, so we assume that external cortisol not only adheres but is incorporated into the hair shaft. Structural damage of older, distal hair segments may facilitate permeability of the hair matrix and diffusion of cortisol from and into aqueous solutions. Thus, the validity of HCC as a marker of stress is compromised in animals where soiling of hair with body fluids is a risk factor. Therefore, hair samples should be collected from clean body regions and, if possible, using proximal hair segments.     

Assaying progesterone,estradiol and cortisol concentrations in hair of Père David deer hinds: an alternative way to reflect seasonality of steroid secretion

The use of a hair hormone concentration assay is increasingly recognized as a useful and noninvasive technique for monitoring the endocrinological status of animals. However, few studies have focused on reproductive and stress hormones together. We used a chemiluminescent immunoassay to determine whether the progesterone, estradiol, and cortisol concentrations could be measured from hair and whether these hormone concentrations varied in different hair segments of captive Père David deer hinds. We found that progesterone, estradiol, and cortisol could be measured in the hair samples and that the progesterone concentration varied but the estradiol and cortisol concentrations did not among different hair segments. Contrary to the segmental decline in hair cortisol found in many studies, we found that progesterone concentration was higher near the tip than at the base of hair in Père David deer. This suggests that the variation in segmental hair steroid hormone concentration in seasonal molting animals may be mainly due to internal reproductive cycles and that hair steroid hormones may reflect long-term physiological changes and can thus be used for the conservation and management of wildlife.     

基于头发蛋白质组学区分个人特质的方法学探究

目的 头发是一类重要的皮肤附属物,主要由角蛋白和角蛋白相关蛋白等组成。不同种族及性别样本的头发蛋白质组成和占比存在差异,且目前缺乏高效率提取头发蛋白的方法。本文探究基于定量头发蛋白质组学方法,旨在探索该方法区分不同个体的可能性。方法 以3例头发样本,对样品处理方法和裂解缓冲液进行探究,发展一种名为PLEE (PTM lab for protein extraction from hair with high efficiency)的稳定、高效的头发蛋白质提取方法,对7例人发样本,以PLEE法进行提取,结合胶内消化方法进行蛋白质组学实验,产出蛋白质组学数据,分析个体间的头发蛋白质组成及占比。结果 共鉴定274种蛋白质,共有的蛋白质107种,非共有蛋白质种类在57～119,部分样本存在独特鉴定蛋白。使用共鉴定107种蛋白质进行定量蛋白质组分析,通过聚类和主成分分析,可将各样本进行区分,且技术重复样本可聚在一起,表明流程的稳定性。另外,筛选出10个关键蛋白（KRT33A、KRTAP9-6、KRT83、KRTAP7-1、KRT32、BLMH、KRT38、KRTAP11-1、NPAS1、KRTA...     

Coat condition, housing condition and measurement of faecal cortisol metabolites--a non-invasive study about alopecia in captive rhesus macaques (Macaca mulatta)

BACKGROUND: Previous studies have characterized alopecia in captive rhesus macaques (Macaca mulatta) by a mixed partial to complete alopecia in a bilateral symmetric pattern. METHODS: In this study, coat condition assessments were related to exogenous and endogenous factors in captive rhesus macaques under different housing conditions in order to identify disturbances in environmental factors controlling or influencing hair growth. Additionally, the degree of alopecia was investigated in relation to adrenal endocrine function as an indicator of social stress using faecal glucocorticoid measurements. RESULTS: Hair loss was found to vary with season and sex, was most pronounced in adult females during the winter and spring months. Generally, infants were not affected, but alopecia developed during adolescence. However, the housing system, available enclosure space and variations in group size and composition also appeared to influence coat condition. Levels of immunoreactive cortisol metabolites (11-oxoetiocholanolone) in faeces were significantly negatively correlated with alopecia, suggesting a relationship between hypothalamic-pituitary-adrenal (HPA) axis activity and hair loss in captive rhesus macaques. CONCLUSIONS: Although the present study demonstrates the influence of the HPA axis on coat condition, it is not known if hair loss is caused by abnormal behaviour or hormonal imbalances of the HPA axis itself. Our data suggest that alopecia in rhesus macaques is a highly complex multicausal disorder.     

Effect of equilibrium pH on the structure and properties of bleach-damaged human hair fibers

Hair proteins are significantly affected by environmental pH. This impact tends to increase with prior hair damage. To understand how pH affects bleached hair properties, we utilized a number of techniques allowing for the determination of hair thermal properties, swelling and water sorption, and dry and wet tensile properties. At pH 5, hair proteins had the best structural integrity, as determined by differential scanning calorimetry and the highest tensile modulus. At pH 10, protein cross-linking density decreased, water content and hair cross-sectional diameter increased. Alkaline treatment, when compared with pH 5, did not reduce intermediate filament conditions (evaluated via enthalpy measurement) nor mechanical property performance in the wet state. In contrast to alkaline-treated hair, bleached hair equilibrated at pH 3 behaved very differently: it contained two different crosslink density zones, was the least stiff in dry and stiffest in wet conditions. Additionally, it absorbed less water and had the lowest diameter because of reduced water binding by protonated carboxyl groups. The pH 3 to 10 did not affect the mechanical strength of bleached hair in dry or wet conditions.     

Evaluation of a method to measure long term cortisol levels

IntroductionElevated levels of cortisol are known to induce various symptoms and diseases, e.g. abdominal obesity, type 2 diabetes, osteoporosis and cardiovascular disease. Measuring serum, saliva and urine cortisol is limited to one time point. Measurement of cortisol in scalp hair is a recently developed method to measure long term cortisol levels. The aim of this study was to investigate whether hair cortisol is a feasible parameter to measure cortisol exposure.ExperimentalWe collected hair samples of 195 healthy individuals, 9 hypercortisolemic and one hypocortisolemic patient and measured hair cortisol levels. Cortisol was extracted from scalp hair using methanol and cortisol levels were measured using a salivary ELISA kit. Measurement of waist and hip circumferences and blood pressure was performed in 46 healthy subjects.ResultsWe found a positive correlation between hair cortisol and both waist circumference (r = 0.392, p = 0.007) and waist-to-hip ratio (WHR) (r = 0.425, p = 0.003). No correlations were found between hair cortisol levels and BMI, blood pressure or age. There was no decline in cortisol levels in six consecutive hair segments. Hair cortisol levels were elevated in patients with known hypercortisolism (p < 0.0001).ConclusionsHair cortisol was positively correlated with WHR, suggesting that hair cortisol reflects cortisol exposure at tissue level, which was also supported by elevated hair cortisol levels in hypercortisolemic patients and concordance between hair cortisol levels and clinical disease course. Cortisol levels in hair are slightly influenced by hair treatment but not by natural hair colour, use of hair products, gender or age.     

Lithium in scalp hair of adults,students, and violent criminals

The lithium content of human hair shows an approximately linear response to extradietary lithium supplementation at dosage levels of up to 2000 μg/d. From the mean hair lithium concentration of 0.063 μg/g in 2648 predominantly American adults, and the reference hair lithium concentrations determined in the present study, the mean lithium intakes were calculated to be 730 μg/d. Hair lithium concentrations were extremely low in nearly 20% of the American samples, and in samples collected in Munich, Germany and Vienna, Austria. Hair lithium levels are low in certain pathological conditions, e.g., heart disease, in learning-disabled subjects, and in incarcerated violent criminals. The highest levels were observed in samples of a lithium-treated psychiatric patient. A statistically highly significant direct association was observed between the hair lithium and cobalt concentrations, which suggests a role of lithium in the transport and distribution of vitamin B₁₂. Interactions of lithium with other trace elements are also discussed.     

Analysis of hair samples of mummies from Semna South (Sudanese Nubia)

Hair samples from 76 burials at Semna South (Sudanese Nubia) were examined using a variety of techniques. Electrophoresis and fluorescence microscopy indicated some oxidation of the cuticule and keratin protein had taken place. However, the cuticular structure and the lack of fluorescence of the cortex indicate that the low humidity and non-alkaline conditions preserved the physical and chemical properties of the hair well. Pigmentation, even allowing for oxidation of melanin, showed a higher proportion of lighter samples than is currently associated with the Nubian area. Hair form analysis showed medium diameter and scale count; the curling variables were intermediate between European and African samples. There was a high ratio of maximum to minimum curvature (a measure of irregularity), approached only by Melanesian samples. Meroitic and X-group burial types were not statistically significantly different (largely due to sample sizes), but the X-group, especially males, showed more African elements than the Meroitic in the curling variables. Principal components analysis showed the Semna sample to be significantly different from seven populations examined earlier.     

Focus: The Science of Stress: Social Fear in US Infants: The Roles of Hair and Salivary Cortisol

Elevated social fear in infancy poses risk for later social maladjustment and psychopathology. Hair cortisol concentration (HCC), an index of cumulative cortisol exposure, and diurnal salivary cortisol slope, a biomarker of acute stress regulation, have been associated with social fear behaviors in childhood; however, no research has addressed their relations in infancy. Elucidating potential biomarkers of infant social fear behaviors, as well as environmental factors associated with these biomarkers, may grant insights into the ontogeny of fear behaviors that increase risk for internalizing and externalizing psychopathologies later in life. The current study used multiple linear regression to examine if infant HCC, infant diurnal cortisol slope, and income-to-needs ratios (ITN) were differentially associated with observed social fear responses to a Stranger Approach task at 12 months. Using a sample of 90 infants (M_age = 12.26m, SD = 0.81m, 50% female), results indicated that increased infant HCC was associated with increased distress vocalizations during the Stranger Approach task, while steeper diurnal cortisol slope was associated with fewer distress vocalizations. Ordinary least squares path analyses did not reveal group differences between economically strained and non-strained infants in how cortisol measures and social fear responses related. Findings underscore very early psychobiological correlates of fearfulness that may increase risk for fear-related disorders and adverse mental health symptomology across childhood.     

Genipa americana L.: A New Phytochemical for White Hair Coloring

This work describes a new hair dyeing methodology using a chemical reaction between geniposide, an iridoid glycoside extracted from the fruit of Genipa americana (geniposide extract, GE) and the amine group of hair keratin. The influence of reaction conditions (pH, temperature, and extract concentration) on the staining of hair fibers, color development, fiber morphology, and mechanical hair properties of black and white human hair samples, was evaluated before and after GE dyeing treatment. Eye contact safety of GE was also studied using HET-CAM. The treatment of white hair fibers using GE at 20 mg mL⁻¹, temperature of 80 °C and pH 5.5 presented the greatest color change (ΔE=54.0). The higher pH influence was observed at pH 10.0 on white hair tresses (ΔE=6.8), using an GE concentration of 20 mg mL⁻¹ and room temperature (25 °C). Treated samples showed marked changes on mechanical and morphological properties. The HET-CAM did not show any change, thus demonstrating that using GE is safe. In conclusion, the temperature and concentration of the extract were the variables that mostly influenced the color and hair damage. A new approach for hair dyeing was established where iridoids may potentially be useful as a natural hair dyeing.     

Integrating cortisol and isotopic analyses of archeological hair: Reconstructing individual experiences of health and stress

Archeological hair from 14 adults from the Nasca Region, Peru (c. AD1–1000) was analyzed for carbon and nitrogen isotopic compositions and cortisol levels. We investigated the relationship between isotopic compositions, which reflect diet, and cortisol, which reflects biogenic cortisol production and chronic stress. Using a case study approach, we determined that there are consistent changes in cortisol production associated with the rapid dietary change characteristic of local mobility. Moreover, changes in nitrogen- and carbon-isotope compositions, when integrated with cortisol levels, enabled inferences to be made about nitrogen metabolism and carbon routing, and elucidated the nature of potential stressors in the months before death. The isotopic and cortisol data suggested a relatively high rate of exposure to stress that is consistent with what is known about the Nasca Region social and physical environments. Of the 14 adults included in this study, six likely suffered from illness/trauma before death, and a further three experienced stress without an observable associated change in isotopic composition. Five individuals also experienced increased stress related to local mobility, inferred from co-occurring changes in cortisol production and dietary shifting. The integration of cortisol and isotopic data revealed individual characteristics of hidden frailty and risk that would not be apparent using more traditional methods of evaluating health status. This approach will provide a powerful enhancement to the understanding of stress, morbidity, and well-being developed through skeletal analysis. Am J Phys Anthropol 156:577–594, 2015. © 2014 Wiley Periodicals, Inc.     

Scales Tell a Story on the Stress History of Fish

Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit α1 of the Na⁺/K⁺-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.     

Using dot blot with immunochemical detection to evaluate global changes in SUMO-2/3 conjugation

Here we describe a non-invasive method for rapid and highly reproducible genotyping of transgenic mammals with ubiquitous expression of fluorophore reporters. Hair samples from transgenic mice and pigs with systemic expression of the fluorophore reporter Venus were analyzed with a fluorescence microscope in few minutes. The hair samples can be preserved for long-term storage at ambient temperature conditions. This non-invasive method is useful for genotyping of transgenic large animals and contributes to animal welfare by reducing stress and discomfort of the animals during sample collection.     

Individual‐based analysis of hair corticosterone reveals factors influencing chronic stress in the American pika

Glucocorticoids are often measured in wildlife to assess physiological responses to environmental or ecological stress. Hair, blood, saliva, or fecal samples are generally used depending on the timescale of the stress response being investigated and species‐specific considerations. Here, we report the first use of hair samples to measure long‐term corticosterone levels in the climate‐sensitive American pika (Ochotona princeps). We validated an immunoassay‐based measurement of corticosterone extracted from hair samples and compared corticosterone estimates obtained from plasma, hair, and fecal samples of nine pikas. To demonstrate an ecological application of this technique, we characterized physiological stress in 49 pikas sampled and released at eight sites along two elevational transects. Microclimate variation was measured at each site using both ambient and subsurface temperature sensors. We used an information theoretic approach to compare support for linear, mixed‐effects models relating corticosterone estimates to microclimate, body size, and sex. Corticosterone was measured accurately in pika hair samples after correcting for the influence of sample mass on corticosterone extraction efficiency. Hair‐ and plasma‐based estimates of corticosterone were weakly correlated. The best‐supported model suggested that corticosterone was lower in larger, male pikas, and at locations with higher ambient temperatures in summer. Our results are consistent with a general negative relationship between body mass and glucocorticoid concentration observed across mammalian species, attributed to the higher mass‐specific metabolic rates of smaller bodied animals. The higher corticosterone levels in female pikas likely reflected the physiological demands of reproduction, as observed in a wide array of mammalian species. Additionally, we establish the first direct physiological evidence for thermal stress in the American pika through nonlethal sampling of corticosterone. Interestingly, our data suggest evidence for cold stress likely induced during the summer molting period. This technique should provide a useful tool to researchers wishing to assess chronic stress in climate‐sensitive mammals.