Diurnal Cortisol Profiles of Stress and Hardiness in Sexual Minority Young Adults. A dissertation presented to. the faculty of

Transcription

1 Diurnal Cortisol Profiles of Stress and Hardiness in Sexual Minority Young Adults A dissertation presented to the faculty of the College of Arts and Science of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Wilson S. Figueroa August Wilson S. Figueroa. All Rights Reserved.

2 This dissertation titled 2 Diurnal Cortisol Profiles of Stress and Hardiness in Sexual Minority Young Adults by WILSON S. FIGUEROA has been approved for the Department of Psychology and the College of Arts and Sciences by Peggy M. Zoccola Associate Professor of Psychology Robert Frank Dean, College of Arts and Sciences

3 Abstract 3 FIGUEROA, WILSON S., Ph.D., August 2017, Experimental Psychology Diurnal Cortisol Profiles of Stress and Hardiness in Sexual Minority Young Adults Director of Dissertation: Peggy M. Zoccola Psychosocial stress, including the experience of and responses to stigma and discrimination, is prevalent in the lives of sexual minorities and may contribute to the existing health disparities between sexual minorities and heterosexual persons. The majority of the research on physical health in sexual minority adults has focused on risky health behaviors (e.g., substance use, unsafe sex behaviors), rather than sources of resilience. Resiliency factors, such as psychological hardiness, may protect against factors that contribute to negative health outcomes, including activation of stress-related physiological systems, such as the hypothalamic-pituitary-adrenal (HPA) axis and its end-product, cortisol. Cortisol is an important stress-related biomarker, and daily cortisol concentrations have been linked to a variety of health outcomes, including cardiovascular disease. The goal of the current study was to test the associations between stress and resiliency factors and daily cortisol concentrations in a sample of 48 sexual minority young adults who were observed over a week-long period of everyday life. The first aim was to test the extent to which sexual minority-specific stressors experienced over the span of a week were associated with diurnal salivary cortisol. The second aim was to examine psychological hardiness as a potential source of resiliency. Additional analyses examined daily general negative and positive events and their relationship to diurnal cortisol. Results indicated that general negative and positive events, but not minorityspecific stressors, were associated with diurnal cortisol in this sample. Further, the

4 4 challenge subcomponent of psychological hardiness was associated with steeper cortisol slope across the week, but total hardiness scores were not. Implications of findings and future directions are discussed.

5 Dedication 5 I would like to dedicate this dissertation to my family and friends for their support throughout my graduate school career. I would also like to dedicate this dissertation to my partner, Mark Yoder, for his love and encouragement.

6 Acknowledgments 6 I would like to thank my adviser, Peggy Zoccola, for her patience, guidance and invaluable feedback not only in this dissertation but throughout my entire graduate career. I would also like to thank my fellow lab mates for all their assistance in collecting, cleaning and analyzing data for this project.

7 7 Table of Contents Page Abstract... 3 Dedication... 5 Acknowledgments... 6 List of Tables... 8 List of Figures... 9 Introduction Models of Stress and Health Daily Diary Methods in Studies of Sexual Minority Stress and Health Psychological Hardiness Aims and Hypotheses Methods Analysis Results Discussion...60 References... 68

8 List of Tables 8 Page Table 1. Bivariate correlations of cortisol, key variables, and covariates...30 Table 2. Characteristics of participants by study arm Table 3. Endorsement of minority stressors. 42 Table 4. Endorsement of negative events Table 5. Endorsement of positive events.56

9 List of Figures 9 Page Figure 1. Flow chart of DASH study from April 2015 to June 30, Figure 2. Average diurnal cortisol slopes Figure 3. Temporal changes in raw salivary cortisol predicted by challenge...50 Figure 4. Total cortisol output predicted by negative events Figure 5. Total cortisol output predicted by positive events Figure 6. Temporal changes in raw salivary cortisol predicted by positive events...59

10 Introduction 10 Minority stress is prevalent in the lives of sexual minorities (Katz-Wise & Hyde, 2012), and associated with poor self-reported health (e.g., Figueroa & Zoccola, 2016). Further, research has shown that everyday stressors may be detrimental to health over time in general populations (for review, see Almeida, 2005). Yet, the mechanisms linking stress to poor health in sexual minorities remain poorly understood. To explore these mechanisms, it is important to examine objective markers that may link stress to poor health. One such marker is the stress hormone cortisol. Cortisol receptors are found throughout the body, allowing cortisol to regulate multiple physiological systems (Hoehn et al., 2010). Thus, identifying how stress processes may influence cortisol has widereaching consequences for sexual minorities. Accordingly, the current study tested the association between minority stress and general negative events and cortisol in the daily life of sexual minority young adults. Additionally, resiliency factors that promote health have been under-examined in sexual minority populations (Herrick et al., 2014). Only preliminary cross-sectional work implies that psychological hardiness, or a personality composite consisting of a sense of control over one s life, commitment to engaging in life and seeing stressors as challenges to overcome, is negatively associated with anxiety, depression, and somatic complaints in sexual minorities (Figueroa & Zoccola, 2015). Thus, the present project also aimed to explore how psychological resilience may and daily positive events influence cortisol in the everyday life of sexual minority adults.

11 Models of Stress and Health 11 Minority Stress Meta-analytic work on minority health suggests that stressors unique to minority populations, or minority stressors, may play an important part in the stress-health association in minority populations (Pascoe & Smart Richman, 2009). The minority stress model of stress and (mental) health (MSM; Meyer, 1995,2003) posits that minority stress may lead to poor health outcomes in minority populations, via two pathways: 1) external processes such as overt discrimination and 2) internal processes such as internalization of negative stereotypes associated with the minority status. Researchers have found that these pathways may lead to poor health both directly and indirectly. Specifically looking at the indirect pathways, it has been found that minority stress may lead to poorer health behaviors (e.g., smoking) as well as heightened or dysregulated physiological stress responses (e.g., exaggerated cortisol output) which in turn may lead to poor health (Pascoe & Smart Richman, 2009). Although minority stress occurs at multiple levels (e.g., systemic discrimination or interpersonal discrimination), minority stress at the interpersonal level is especially prevalent and consistently associated with self-reported poor health (e.g., Figueroa & Zoccola, 2016; Wise & Hyde, 2012). For example, a recent meta-analysis of 164 studies from examining lifetime victimization experiences of sexual minority individuals found that 55% of sexual minority men and women experienced verbal harassment, 41% experienced discrimination, and 45% experienced sexual harassment due to their sexual orientation (Katz-Wise & Hyde, 2012). Similarly, in a seven-day paper and pencil diary study of 69 sexual minority adults (51% women, 86% Caucasian;

12 Mage = 31), participants reported an average of two heterosexist hassles (e.g., 12 discrimination due to sexual orientation) across the week with a range of 0 to 8 events (Swim, Pearson, & Johnston, 2007). Thus, minority stress appears to be prevalent in the lifetime as well as everyday lives of sexual minority adults. Minority Stressors and Health To date, only a few studies have tested the association between minority stress and self-reported physical health (e.g., Figueroa & Zoccola, 2016; Frost, Lehavot & Meyer, 2013). For example, in an online sample of 277 sexual minority adults, greater perceived discrimination from family and friends was associated with greater somatic complaints (e.g., headaches), and poorer general physical health, controlling for general stressors (Figueroa & Zoccola, 2016). Although the literature examining minority stress and physical health is limited, it appears that there is a direct association between minority stress and poor (self-reported) physical health in sexual minorities. Nevertheless, to the author s knowledge, the association between minority stress and physical health (subjective or objective) in sexual minorities has not been examined in daily life. Physiological stress responses to minority stress are proposed to mediate the association between minority stress and poor health in sexual minorities (e.g., Pascoe & Smart Richman, 2009). Previous work has found that perceived stress reactivity (i.e., a subjective measure of stress reactivity in a variety of different domains) partially mediated the association between perceived discrimination and self-reported health outcomes (Figueroa & Zoccola, 2016). Yet, in order to fully examine the pathways between minority stress and health, researchers must also examine objective

13 physiological measures of stress responses as they may be important mediators in the 13 association between stress and health in sexual minority individuals. Stress, Cortisol, and Health One such possible biological mediator is cortisol. Cortisol, a glucocorticoid hormone synthesized from cholesterol in the adrenal cortex, and the primary end-product of the Hypothalamic-Pituitary-Adrenal (HPA) axis, is released in response to psychosocial stress (Kirschbaum & Hellhammer, 1989). A psychosocial stressor can trigger the HPA axis to release a cascade of hormones with the eventual release of cortisol (Kemeny, 2003). The primary functions of cortisol are to increase blood sugar through gluconeogenesis (i.e., a mechanism by which blood glucose levels are maintained in the human body), suppression of the immune system, and aiding of the metabolism of fat, protein, and carbohydrates (Hoehn et al., 2010). Cortisol receptors are located throughout the entire body, therefore cortisol has the ability to widely act upon tissue in the body (e.g., preparing the body physiologically to respond to stressors; Charmandari, Tsigos, & Chrousos, 2005). Cortisol normally follows a stable biological rhythm throughout the day. In general, cortisol typically rises sharply in the morning upon awakening (the rise in cortisol that occurs minutes after awakening is known as the cortisol awakening response or CAR) then steadily declines throughout the day (cortisol slope; Wust et al., 2000). Cortisol can also be measured by assessing daily output, or total exposure to cortisol throughout the day, which is usually calculated as the area between ground and cortisol values taken throughout the day (area-under-the-curve, with respect to ground: AUCG). Flat or blunted CAR responses, cortisol that does not decline throughout the day,

14 14 and persistently high total daily cortisol output may be indicative of dysregulation due to long-term response to chronic stress (McEwen, 1998). In the short term, cortisol responses to psychosocial stressors are adaptive and essential to stress responses (e.g., Steptoe, Hamer, & Chida, 2007). However, chronic over (or under) activation of the HPA axis has been implicated in a variety of different disorders such as depression and autoimmune disease (Chrousos & Gold, 1992). For example, repeated exposure to discrimination (e.g., harassment, violence, and victimization) is thought to lead to heightened stress reactivity through dysregulation of physiological systems (Myers, 2009). Repeated exaggerated stress reactivity may ultimately lead to dysregulation of physiological systems (McEwen, 2004) as well as interfere with the body s restorative processes (e.g., sleep and relaxation; Beatty et al., 2011). Failure to return to baseline levels following a stressor, or sustained cortisol elevations, may indicate an impairment of restorative processes that, when impaired, are linked to disease risk and mortality (e.g., cardiovascular disease; Gerin & Pickering, 1995; depression; Burke, Davis, Otte, & Mohr, 2005). Therefore, it is important to examine biological mediators like cortisol because doing so may elucidate why minority stress is associated with poorer health in sexual minorities. Sexual Minority Stress and Cortisol In total, only three studies have specifically examined the association between minority stress and cortisol output among sexual minority individuals (Burton, Bonanno, & Hatzenbuehler, 2014; Hatzenbuehler & McLaughlin, 2013; Parra, Benibgui, Helm, & Hastings, 2016). Parra and colleagues (2016) found that recent major LGBT-related stressors were significantly positively related to flatter diurnal cortisol slopes and

15 depressive symptoms, and flatter diurnal cortisol slopes statistically mediated the 15 association between recent LGBT- related stress and depressive symptoms. The current study adds to the study by Parra and colleagues by examining daily minority stressors as well as measuring diurnal cortisol in more nuanced fashion (i.e., examining diurnal cortisol over seven days compared to only one day). With regards to cortisol reactivity, two recent studies found that less familial support (Burton et al., 2014) and growing up in areas with high structural stigma (e.g., or discrimination at the societal level such as lack of employment non-discrimination policies based on sexual orientation, presence of constitutional amendments banning same-sex marriage; Hatzenbeuhler & McLaughlin, 2014) were associated with cortisol reactivity in response to a speech stressor in sexual minorities. Specifically, less familial support was associated with greater cortisol reactivity and higher structural stigma was associated with blunted cortisol responses to the speech stressors. Exaggerated cortisol reactivity may indicate HPA axis dysregulation and has been associated with poor health outcomes (e.g., cardiovascular disease; Chrousos, 2009; Kirschbaum et al., 1995). Further, as stated previously, non-normative cortisol trajectories (e.g., blunted cortisol responses) are associated with long-term health consequences (e.g., heart disease; Matthews et al., 2006). Overall, only a few studies have examined cortisol responses in the sexual minority population. Preliminary evidence suggests the presence of non-normative cortisol (e.g., flatter slopes, blunted reactivity) in sexual minority individuals who grew up in areas high in structural stigma or experienced greater minority stressors in recent history. There is also initial evidence for greater cortisol reactivity for those with less

16 familial support. Nevertheless, the association between daily experiences of minority 16 stress and diurnal cortisol has yet to be examined in sexual minorities.

17 Daily Diary Methods in Studies of Sexual Minority Stress and Health 17 To date, the majority of studies that have examined the associations between stress, physical health, and stress responses in sexual minorities have been limited to cross-sectional designs and self-report measures. In contrast, daily diary designs allow for increased ecological validity by assessing events as they unfold, reducing the retrospection bias that is often associated with cross-sectional methods at a single timepoint. Further, daily diary studies can examine how daily events are associated with changes in well-being from one day to the next, including changes in physiological measures such as salivary cortisol. Further, recent findings suggest that stressors, hassles, and challenges experienced in everyday life, such as arguments with loved ones, may be more detrimental to health in the long-term than time-limited major life events. In a study of 216 adults it was found that daily hassles were associated with poorer overall health (e.g., chronic conditions such as hypertension), independent of major life stressors (e.g., seriously personal injury) (Almeida, 2005). More specifically, daily hassles explained an additional 9% of the variance in overall poor health after controlling for major life stressors. Given that daily stressors are associated with immediate psychological and physiological responses, the compounded effects of daily stressors may be even more detrimental to health than less frequent major life stressors. It is therefore especially important to examine daily stressors and associated psychological and physiological changes in at-risk populations such as sexual minority young adults. To date, daily hassles have been under examined in sexual minority samples.

18 Psychological Hardiness 18 Although variables that may confer risk to health outcomes in sexual minorities are important to examine, variables that may be beneficial to health outcome should be studied as well. Psychological hardiness is a personality composite consisting of control (having a sense of control over life events), commitment (being engaged and having genuine interest in life), and challenge (seeing stressors as opportunities for growth; Maddi, 2007). Hardy individuals are optimistic, and readily able to find positive meaning in life (Kobasa, 1979). Psychological hardiness has been shown to be associated with better self-reported mental and physical health in non-sexual minority populations (Taylor, Pietrobon, Taverniers, Leon, & Fern, 2013). Independent of stress, individuals high in psychological hardiness have better mental and physical health because they are more willing to engage in health-promoting activities such as maintaining a healthy diet and exercise (Maddi, 2007). Although hardiness is considered a personality factor, individuals are able to increase psychological hardiness via training (Khoshaba & Maddi, 2001). Hardiness may also influence the relationship between stressful life events and health outcomes (e.g., Dion, Dion, & Pak, 1992). In the face of stressors, hardiness is typically found to buffer against the effects of general stress (i.e., not minority specific stress) on health. For example, Sandvik and colleagues (2013) found that Norwegian navy cadets with greater hardiness scores were less physiologically reactive to intense naval training with regards to immune and neuroendocrine responses compared to those who had low hardiness scores. In addition, hardiness was found to buffer the association between self-reported discrimination and self-reported health (Dion et al., 1992). In this

19 study, psychological health was assessed via a composite that examined nervousness, 19 sleep problems, headaches, inability to cope, typical mood, and degree of worrying. Dion and colleagues (1992) found that, among Chinese immigrants living in Toronto who reported low hardiness, there was a moderate association between self-reported discrimination and poor psychological health. In contrast, the association between discrimination and psychological health was not significant among participants high in hardiness. The potential moderating role of hardiness in the association between minority stress and biological stress responses or diurnal cortisol has not been examined. However, past work examining sexual minority adults in the U.S. found that hardiness moderated the association between stigma consciousness and self-reported physical health (Figueroa & Zoccola, 2015). In an online sample of 277 sexual minority adults (44% female), it was found that the association between stigma-consciousness and self-reported physical symptoms (e.g., back aches, headaches, etc.) was dependent on the level of hardiness reported; greater hardiness levels weakened the association between stigmaconsciousness and self-reported physical symptoms. It was also found that there was a direct negative association between hardiness and poor self-reported mental and physical health as well as self-reported physical symptoms, indicating better self-reported health. The current study built on and expanded this work by examining the direct and moderating effect of psychological hardiness on diurnal cortisol.

20 Aims and Hypotheses 20 The first aim was to examine how sexual minority-specific stressors experienced over the span of a week are associated with diurnal salivary cortisol in sexual minority young adults. It was hypothesized that cortisol output across the week would be greater for those who self-report greater sexual minority stressors compared to those who report less stressors (Hypothesis 1). The second aim of the current study was to examine psychological hardiness as a potential source of resiliency in sexual minority individuals. It was hypothesized that: (a) there would be a direct association between hardiness and cortisol output, such that greater hardiness would be associated with lower cortisol output across the week (Hypothesis 2a). It was further hypothesized that (b) there would be an interaction between hardiness and daily sexual minority stressors (averaged across the week) and cortisol output across the week, such that greater hardiness would weaken the association between daily stressors averaged across the week and cortisol output across the week (Hypothesis 2b). The subcomponents of hardiness (commitment, control and challenge) and their association with diurnal cortisol were also explored. As an exploratory aim, the association between stress, resiliency and diurnal cortisol slope was also examined. Drawing from recent studies, the current study tested whether greater daily sexual minority stressors were associated with flatter diurnal cortisol slopes (Hypothesis 3), and whether the association between daily stressors and cortisol diurnal slope was dependent on hardiness levels, such that greater hardiness would be associated with steeper trajectories (Hypothesis 4). Finally, the association between daily negative and positive events and diurnal cortisol were also explored.

21 Methods 21 As seen in the Figure 1, one-hundred and twenty-one (n = 63 survey only, n = 58 survey plus cortisol) sexual minority young adults were enrolled in a weeklong study in which they were asked to complete Internet-based evening surveys as well as provide four daily saliva samples to assess diurnal cortisol. As few exclusion criteria as possible were used to maximize generalizability and reduce confounding of the cortisol measures. Participants were considered ineligible for the current study if they indicated the following at the initial screening: (1) they were not between the ages of 18 and 35, (2) they identified as both heterosexual and cisgender, (3) they did not have reliable Internet access between 9:00 PM and 1:00 AM in order to successfully comply with the study s protocol, (4) they were pregnant, (5) they used steroid-based medication, or (6) they reported having a major psychiatric or endocrine disorder (e.g., de Weerth & Buitelaar, 2005; Burke, Davis, Otte, & Mohr, 2005; Granger, Hibel, Fortunato, & Kapelewski, 2009). Recruitment A flexible recruitment approach to the identification of sexual minority participants was taken by using snowball sampling methods that have been successfully applied in a variety of difficult-to-reach samples, including sexual minorities (Browne, 2005). With the assistance of the director of Ohio University s LGBT center, LGBT+ organizations throughout Ohio and the surrounding Midwest region were contacted to aid in recruitment. lists, personal contacts of the research team and social media (e.g., Facebook) were also used to recruit potential participants. Participants were also encouraged to send recruitment links to any contacts who may have been eligible.

22 Figure 1. Flow chart of Daily Activities, Stress, & Health: DASH Study study from April 2015 to June 30,

23 Procedure 23 Interested individuals were screened for eligibility via an online survey that was linked to recruitment communications. As seen in Figure 1, eligible participants (n = 188) were then invited to complete a weeklong study of Daily Activities, Stress, & Health: DASH Study. Initial screening survey contained items assessing demographic variables as well as the eligibility criteria previous mentioned. If participants were eligible, they were then linked to an online consent form for the baseline survey. After reading the online consent and agreeing to participate in the study, participants were then taken to the baseline survey (which assessed variables that have been found to be associated with cortisol, such as health behaviors, as well as basic demographic information, psychological hardiness, social support, and general stress). The survey took approximately 30 minutes to complete, but participants were given up to 90 minutes to complete the survey. Individuals who successfully completed the baseline survey (n = 134) were invited and consented to participate in the weeklong portion of the study. Randomizer.org was used to generate blocked allocation sequences to randomly assign participants to one of two arms of a larger study: 1) complete evening surveys only or 2) complete evening surveys plus cortisol sampling. Consent was obtained via phone and paper version that was returned when the participant had completed the study. The current study focused on the participants assigned to the evening survey plus cortisol condition (n = 58). For all study participants, the weeklong portion of the study consisted of seven nightly surveys that assessed occurrences of sexual minority-specific stressors, negative and positive events, perceived stress and additional variables that may be associated with

24 24 cortisol (e.g., sleep/wake time). Participants were allowed to complete the daily evening survey from 9:00 PM to 1:00 AM in order to minimize variability in response time. Daily evening surveys took approximately minutes to complete, but the participant had up to 60 minutes to complete the survey before the survey link became inactive. In the survey plus cortisol sampling arm of the study (i.e., the focus of the present investigation), the weeklong study also consisted of saliva collection across the seven days. Diurnal cortisol was assessed via four time points of saliva collection: (1) upon awakening, (2) 45-minutes after awakening, (3) 12 hours after awakening, and (4) bedtime. These collection time points were selected to capture diurnal cortisol slope and output (MacArthur Research Network on Socioeconomic Status and Health, 2000). Although participants were sent and text reminders, they were also encouraged to set their own alarms on their phone to aid with compliance. After the participant was consented over the phone, a saliva package was prepared and shipped to the participant. The saliva package contained the following: 1) paper version of consent form, 2) seven bags of plastic, two-inch straws and tubes to collect saliva, 3) written instructions on how to collect saliva using the straws and tubes, 4) seven paper logs that assess sleep/wake time, food-intake, physical activity, and general stress for the day, 5) a pre-paid return mailer, and 6) information on nearby package drop off points to return the saliva package after the last day of the study. Participants were compensated $15 (via electronic store gift card) for completing the baseline survey. Participants were compensated $5 for each daily evening survey they completed and an additional $5 for each day of saliva samples they successfully completed, with an additional $5 bonus for completing all evening surveys and a $5

25 25 bonus for completing all saliva samples. In total, participants could receive up to $95 (via electronic store gift card) for participating fully in the study. Of the 58 participants who enrolled in the survey plus cortisol arm of the study, 53 returned saliva samples with a completion rate of 88.6% (1,439 of 1,624 samples) and a completion rate of 86.7% for the daily dairy questionnaires (this rate was slightly lower in the survey only condition, 82.1%, but not statistically different). Key Measures Cortisol. Participants were asked to provide four daily saliva samples across seven days. Participants were instructed to avoid eating, drinking (except water), smoking, exercising, and teeth-brushing until the first two samples are collected, and are urged not to eat, drink, exercise, or brush teeth for one hour before collection of evening samples to avoid sample contamination. Participants stored samples in home freezers until they mailed them back to the laboratory in pre-paid return packaging. It is important to note that cortisol levels are stable at room temperature for several weeks (Clements & Parker, 1998). Returned samples were kept frozen at -80 C until assayed by the research team. Cortisol concentrations for each sample were determined in duplicate and averaged using enzyme-linked immunoassays (ELISA; R&D Systems, Inc.) with standardized procedures. The lower limit of detection was 0.07 ng/ml; intra- and inter-assay variation had coefficients of covariance at less than 10%. The main cortisol outcome of the current study was total exposure or AUCg, and slope was examined as an exploratory aim as the current study may be underpowered to test between person differences (Segerstrom, Boggero, Smith, & Sephton, 2014). Total cortisol output was calculated using the four raw values sampled throughout the day with

26 26 the standard trapezoidal formula (Pruessner, Kirschbaum, Meinlschmid, & Hellhammer, 2003), which results in a value that represents total exposure to cortisol throughout the day (Stawski et al., 2013; Zeiders, Doane, & Roosa, 2012). Daily stressors and events. Sexual minority stressors. An events checklist assessed participants daily experience of sexual minority-related stressors. Twenty sexual minority related stressors were derived from the Gender Expression, Vigilance, Discrimination/Harassment, Vicarious Trauma and Isolation components of the Daily Heterosexist Experiences Questionnaire (Balsam, Beadnell, & Molina, 2013). The scale showed good internal consistency throughout the week with Cronbach alpha s ranging from.74 to.79. Sample items included, Watching what you say and do around heterosexual people, and Being verbally harassed by strangers because you are LGBT. Participants were asked to indicate stressor/event occurrence in the prior 24 hours and degree of emotional impact (e.g., distress). Additional stressors reported by the participants were coded to determine whether or not they were minority-stressor related and if they were they were included in the independent variable of interest in the current study. The primary independent variable in the current study was self-reported exposure to daily minority-stressors. Several coding schemes were explored given the infrequency of daily minority stressors reported. The first coding scheme used was: not at all = 0, rarely = 1, a few = 3, several = 5, and often = 7. Daily sexual minority stressors were totaled for each participant each day and the daily average for each participant were calculated across the week. The second coding scheme used was a binary 0 = none at all (each day) and 1 = any minority stress (each day) and averaged across the week. The third coding scheme

27 used was to combine the first coding scheme with the emotional responses (scored 0 = 27 not applicable to 4 = a great deal of impact) to daily minority stressors. This was done by taking the original coding scheme (i.e., not at all = 0, rarely = 1, a few = 3, several = 5, and often = 7) and multiplying it to the endorsement of emotional response (e.g., if a person endorsed rarely experiencing the stressor, but it had a great deal of emotional impact that would be scored as 1 x 4 or 4). The final coding scheme used was to create an average of the three subcomponents, of the original seven subcomponents of the daily minority stress measure, with the highest endorsement: Isolation, Vicarious Trauma, and Vigilance. Due to the positive skew of the data, the natural log of daily average minority stress of this coding scheme was used. Daily negative events. 10 additional negative events (e.g., financial concerns) derived from the Survey of Recent Life Experience (Kohn & Macdonald, 1992) were examined. The scale showed good internal consistency throughout the week with Cronbach alpha s ranging from.78 to.81. The coding scheme used was not at all = 0, rarely = 1, a few = 3, several = 5, and often = 7. Daily positive events. 10 additional positive events (e.g., socializing with friends) derived from Gable et al. (2000) was also assessed. The scale showed good internal consistency throughout the week with Cronbach alpha s ranging from.80 to.82. The coding scheme used was not at all = 0, rarely = 1, a few = 3, several = 5, and often = 7. Hardiness. Dispositional Resiliency Scale (DRS-15; Bartone, 1991), was used to measure psychological hardiness. The 15-item scale has three facets: commitment, =.81, control, =.73, and challenge, =.71, as well as an overall score, =.80, with the overall score having a range from 0 to 45, with greater scores indicating greater

28 hardiness. The overall score was used to examine Aim 2 and the three facets of 28 psychological hardiness were examined via additional analyses. Additional descriptive variables and covariates. Factors that may influence diurnal cortisol were assessed during the baseline survey, evening surveys, and along with saliva sampling. Such variables include: sex, age, medication use (averaged across the week), typical tobacco use (averaged across the week), caffeine (averaged across the week), and alcohol use (averaged across the week), typical physical activity (averaged across the week), general stress (averaged across the week), depressive symptoms, and sleep/wake times (averaged across the week), (e.g., Gilbert, Dibb, Plath, & Hiyane, 2000; Larsson, Gullberg, Rastam, & Lindblad, 2009; Leproult, Copinschi, Buxton, & Van Cauter, 1997; Pruessner, Hellhammer, Pruessner, & Lupien, 2003; Rask, Olsson, Söderberg, Andrew, Livingstone, Johnson, & Walker, 2001; Supnicki & Obminski, 1992; Thayer, Hall, Sollers, & Fischer, 2006). In order to characterize the sample, the following demographic information was assessed: age, sex, education, income, employment, race/ethnicity, and degree and time of sexual and/or gender identity disclosure, or Outness. Sexual orientation and transgender status are assessed with two items from the 2012 Massachusetts Behavioral Risk Factor Surveillance System Survey. General social support variables (tangible, belonging, and appraisal) were also examined as an additional resiliency measure. Although these variables were correlated with daily events (see Table 1), none predicted diurnal cortisol nor moderated the association between daily events and diurnal cortisol. Therefore, further analyses were not performed.

29 Of the above descriptive variables and covariates, the following were 29 significantly associated with diurnal cortisol (output) and therefore included in analyses: Outness, time of disclosure, typical alcohol and tobacco use, typical physical activity, and wake time (see Table 1).

30 Table 1 30 Bivariate Correlations of Cortisol, Key Variables and Covariates Column Cortisol.09.13* -.17** * -.15* -.13* * -.25* -.27** -.16* Minority.47** ** * *.03.21**.26** -.17* Stress 3. Negative ** -.21** **.21** * Event 4. Positive.16** *.12*.09.12* ** -.15* * ** Event 5. Hardiness.07.28**.38**.14* ** -.65**.15* -.21* -.22* -.51**.15* ** 6. Gender * ** Tangible.78**.55**.22* ** -.17* *.27* ** Support 8. Belonging.63**.25* ** -.37** ** Support 9. Appraisal.35** ** -.15* * Support 10. Outness * -.11*.26* * Disclosure ** *.05.23*.13*.12* Time

31 Table 1: continued 31 Column Depressed.68** * *.15*.05.63** Mood 13. Anxiety * * -.32**.18*.09.67** 14. Age * -.32** ** Caffeine.48** ** Alcohol.35** -.33** Tobacco.08.32**.52**.26** 18. Activity * 19. Wake time.41**.29** 20. Sleep Time.19* 21. General Stress Note. *p <.05, **p <.01; cortisol represents the grand mean across all time points; total disclosure time was calculated by subtracting current age from age of disclosure with higher scores indicating longer time since disclosure; higher Outness scores represent greater degree of total identity disclosure; higher scores in depressed mood, anxiety and the support scales represent greater depressed mood, anxiety and support, respectively; gender coded 0 = female, 1 = male; negative and positive events, general stress, wake time, sleep time, minority stress, typical alcohol, tobacco, caffeine use and activity level are averaged across the week.

32 Analysis 32 Preliminary Analyses Data was examined for variable distribution and outliers with statistical and graphical methods. Of the 53 participants who returned saliva samples, it was found that 5 participants had biological unreasonable or invalid (e.g., values were too high) data. Transgender individuals were not included in the final sample due to the influence of steroid based medication on diurnal cortisol. Therefore, the final sample used for analyses was n = 48. Cortisol values were log transformed due to the natural skew of the data. Primary and exploratory aims were examined using multilevel modeling procedures with the SAS PROC MIXED procedure. Multilevel modeling techniques allow one to model both within- subject and between-subject differences in the overall diurnal cortisol output and response patterns. In other words, it takes into account the non-independent nature of the data. All models were re-analyzed with the significant covariates previously mentioned. Given that saliva collection took place across the week, day of week (weekday = 0; weekend = 1) was also included as a possible covariate. Variables were group mean- or person mean-centered, as appropriate. Analysis for Hypothesis 1 The first aim of the current study was to examine the relationship between daily minority stressors and diurnal cortisol over the span of a week in a sample of sexual minority young adults. The following analyses were also used to examine the association between negative and positive events and diurnal cortisol. A two-level model, where the variability of cortisol measurement within a person as modeled at the level one and the

33 33 inter-individual differences in the physiological response was modeled at the level two, was used. The focus of the current study was on total cortisol output (AUCG), which was measured using the raw cortisol values for all daily samples, with respect to ground (Preussner et al., 2003). Level 1 (day-level): AUC id = α 0i + e id (1a) Level 2 (person-level): α 0i = β 00 + β 01 (Stress i ) + u 0i (1b) The Level 1 outcome variable, AUC id, refers to the cortisol output for person i on day d as a function of a person-specific intercept (α0i), which reflects an individual s average daily AUC. eid is the day-level residual. At level two, the level one coefficient is modeled as a function of the sample s average AUC cortisol (β00) and average daily minority stressor experience (β01), as well as random effects allowing for variation in the AUC (u0i). Furthermore, β01 reflects the association between individual differences in minority stressor frequency and AUC. Analysis for Aim 2 The second aim of the proposed study examined the potential direct and moderating effects on psychological hardiness on diurnal cortisol in sexual minority young adults. Multilevel modeling analyses were used to examine the direct association between psychological hardiness and cortisol output across the week (hypothesis 2a). The equations above were used with the addition of hardiness at a level two-interaction term (interaction with minority stress; hypothesis 2b). Lower order interaction terms were also included. Level 1 (day-level): AUC id = α 0i + e id (1a)

34 34 Level 2 (person-level): α 0i = β 00 + β 01 (Stress i ) + β 02 (Hardiness i ) + β 03 (Stress i * Hardiness i ) + u 0i (1b) In this model β00 is the sample s average of AUC cortisol and β01, β02 and β03 are the effects of average daily minority stressor experience, hardiness and their interactions, respectively, on the sample s average of AUC cortisol. Exploratory Analyses To examine hypotheses 3 and 4 regarding cortisol slope, a three-level model was used. The following analyses were also used to examine the association between negative and positive events and cortisol slope. A level-one submodel represents individual daily change in cortisol as a function of time (across time points within a day). The level-two model represents day-to-day differences within individuals physiological response and a level-three submodel represents inter-individual differences in the physiological response trajectories (across persons, or betweenperson variation). To test hypothesis 3 (cortisol slope as a function of self-reported exposure to minority stress), the following basic three-level model were used: Level 1 (occasion-level): Cortisolido = α0id + α1id (time)id + α2id (time 2 )id + α3id (time 3 )id + eido Level 2 (day-level): α0id = β00i + u0id α1id = β10i + u1id α2id = β20i + u1id α3id = β30i + u1id Level 3 (person-level): β00i = γ000 + γ001(stressi) + v00i β10i = γ100 + γ101(stressi) + v10i β20i = γ200 + γ201(stressi) + v10i β30i = γ300 + γ301(stressi) + v10i

35 At level 1, the equation serves as the daily growth curve model for the diurnal 35 cortisol. Cortisol on occasion o on day d for individual i is a function of an intercept (α0id), a linear slope (α1id), a quadratic slope (α2id) and a cubic slope (α3id) which models the cubic change in cortisol across samples 2, 3 and 4 per hour since awakening (sample 1) and the residual (eido). At level 2, the level 1 intercept and slopes become the outcomes and reflect an individual s average awakening cortisol level (β00i) and an individual s average linear, quadratic and cubic slopes (β10i, β20i and β30i). u0id, u1id, u2id and u3id are the random effects allowing the intercept and slopes to vary within-person across days. Finally, at level 3, the level 2 intercepts become the outcome and reflect the sample average awaking cortisol level (γ000) and slopes (γ100, γ200 and γ300). γ001, γ101, γ201 and γ301 reflect the effect of individual average daily stressor frequency on the awakening cortisol level and the linear, quadratic and cubic slopes, respectively. v00i, v10i, v20i and v30i are the random effects allowing the intercept and slopes to vary across persons. Hypothesis 4 was examined via the above equation with the addition of hardiness and the hardiness by stress interaction term at level 3. The full model is also presented below. Level 1 (occasion-level): Cortisolido = α0id + α1id (time)id + α2id (time 2 )id + α3id (time 3 )id + eido Level 2 (day-level): α0id = β00i + u0id α1id = β10i + u1id α2id = β20i + u1id α3id = β30i + u1id Level 3 (person-level): (Stressi * β00i = γ000 + γ001(stressi) + γ002 (Hardinessi) + γ003

36 36 Hardinessi)+ v00i β10i = γ100 + γ101(stressi) + γ102 (Hardiness) + γ103 (Hardinessi * Stressi) + v10i β20i = γ200 + γ201(stressi) + γ202 (Hardiness) + γ203 (Hardinessi * Stressi) + v10i β30i = γ300 + γ301(stressi) + γ302 (Hardiness) + γ303 (Hardinessi * Stressi) + v10i In this model, γ000 and γ100 are the sample s average awakening cortisol level and sample s average slope respectively. While γ001, γ002 and γ003 reflects the effects of average daily minority stressor experience, hardiness and their interactions, respectively, on the sample s average awakening cortisol level respectively; and γ101, γ102 and γ103 reflects the effects of average daily minority stressor experience, hardiness and their interactions, respectively, on the sample s average. Power Considerations The primary aim of the current study was to examine the effect of daily minority stressors on seven days of salivary cortisol output. Prior simulation research on power in similar two-level multilevel models (Bell, Morgan, Schoeneberger, Kromrey, & Ferron, 2014; Maas & Hox, 2005; Scherbaum & Ferreter, 2009) and related empirical analyses on diurnal stress and cortisol (Hill Golden et al., 2014; Segerstrom et al., 2014) indicate that a minimum sample size of 30 is needed examine fixed-effects parameters. Our sample size of 48 was sufficient to detect moderate to large effect sizes for our hypothesized fixed-effects parameters. Importantly, the inclusion of significant covariates in statistical models helps account for error variance, and thereby increase the proportion of variance in key predictors and statistical power.

37 Results 37 Sample Characteristics Sample characteristics for both arms of the study can be seen in Table 2. As shown in the table, both arms of the study were similar in tested variables except for perceived stress which was higher in the survey plus cortisol arm. The following describes the survey plus cortisol arm of the study, which is the focus of this project. The majority of the participants were female (55.2%), young (Mage = 25.02), white, well-educated and of lower income status. About 47 percent of the participants were full-time students and the majority of the participants were also employed full-time (62.1%). The majority of the participants were also either gay (36.2%) or lesbian (27.6%) and non-transgender (81%). With regards to degree of outness, or the degree to which an individual had disclosed their sexual orientation to different individuals in their lives, on average participants were definitely out, but rarely talked about their orientation (M = 5.17, where 1 = person does not know about your sexual orientation to 7 = person definitely knows your sexual orientation and openly talks about it). Participants had disclosed their orientation to different individuals in their lives (e.g., family and friends) an average age of about 21.40, which equates to approximately 3.6 years before the current study. On average, participants reported relatively little lifetime discrimination (M =.95, where 0 = has never happened and 5 = has happened almost all the time) or internalized homophobia (M = 8.75, range 5-25). Depressed mood (M = 10.67, range 0-60) and anxiety (M = 21.44, range 0-60) were moderately low. In contrast, perceived general stress (M = 2.84, where 0 = never and 4 = very often) and stigma consciousness (i.e., being conscious about negative stereotypes associated with the minority identity; M

38 38 = 4.31, range 0-6 with higher scores indicating greater stigma consciousness) were fairly high. Forty percent (n = 23) reported taking medication (3.4% were on sex hormones, and these individuals were excluded from analyses due to irregular cortisol values; other medication included: Prilosec, Focalin, Truvada, and Clobetasol) and 15.5% (n = 9) reported a chronic health condition (e.g., asthma, hypertension, psoriasis). Twelve percent of the participants reported being smokers (Mdaily = 4.26, SD = 3.09) and 53.1% of the participants reported drinking at least 1 alcoholic drink a week. Overall, participants were fairly inactive, and reported an average of 21.4 (SD = 15.42) minutes of exercise across the week

39 Table 2 39

40 Table 2: continued 40 Cortisol (n = 58) Survey Only (n = 63) M SD Range M SD Range Age (18-35) (18-35) General Stress 2.84*.70 (0-4) (0-4) Outness (1-8) (1-8) Lifetime (0-4) (0-4) Discrimination Stigma (0-6) (0-6) Consciousness Internalized (5-25) (5-25) Homophobia Depressed Mood (0-60) (0-60) Anxiety (0-60) (0-60) Hardiness (0-45) (0-45) Note. *t(119) = 2.22, p =.03

41 Daily Minority Stressors 41 As shown in Table 3, on average, daily minority stress items were infrequently endorsed or not endorsed at all across the week (the total number of minority stress experiences across the week for the average person was about 15). Specifically, it appears that participants reported little to no minority stress when it was directed towards themselves (e.g., being treated unfairly at stores or restaurants, verbal harassment). This was particularly true with regards to minority stress directed towards gender expression, which was mostly not endorsed at all. The most commonly endorsed minority stressor(s) were vicarious crime such as hearing about crimes against people you don t know (5.9%) and isolation having few people to talk to about being LGBT (8.9%).

42 Table 3 42 Endorsement of minority stressor by day for survey plus cortisol arm of study (N = 58) Feeling invisible due to gender expression Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 5 (9.4%) M =.40 SD = (11.5%) M =.15 SD =.50 4 (8.7%) M =.12 SD =.47 4 (8.7%) M =.13 SD =.50 4 (8.5%) M =. 17 SD =.76 5 (10.6%) M =.15 SD=.52 4 (8.3%) M =.21 SD =.85 Daily Sum Averaged Across Week (2.5%) Max = 21 Don t fit in the LGBT community because of gender expression Misunderstoo d due to gender expression Harassed in public because of gender expression Pretending to have opposite sex partner Pretending you are heterosexual 6 (11.3%) M =.34 SD = (11.3%) M =.19 SD =.62 0 M =.00 SD = 0 2 (3.8%) M =.04 SD =.19 9 (17%) M =.28 SD = (19.2%) M =.31 SD =.75 8 (15.4%) M =.23 SD =.64 0 M =.00 SD = 0 1 (2%) M =.02 SD =.14 4 (7.7%) M =.12 SD =.47 3 (5.9%) M =.10 SD =.46 2 (4%) M =.04 SD =.20 0 M =.00 SD = 0 2 (3.9%) M =.08 SD =.44 4 (7.8%) M =.20 SD =.82 6 (13%) M =.17 SD =.53 5 (10.9%) M =.15 SD =.51 1 (2%) M =.02 SD =.15 5 (10.9%) M =.20 SD =.79 7 (15.5%) M =.24 SD =.80 4 (8.5%) M =.21 SD =.86 5 (10.9%) M =.23 SD =.76 0 M =.00 SD = 0 3 (6.4%) M =.15 SD =.75 5 (10.6%) M =.28 SD = (10.6%) M =.19 SD =.65 5 (10.9%) M =.23 SD =.86 1 (2%) M =.02 SD =.15 4 (8.5%) M =.17 SD =.76 7 (14.9%) M =.23 SD =.79 6 (12.5%) M =.17 SD =.52 4 (8.3%) M =.17 SD =.63 0 M =.00 SD = 0 3 (6.3%) M =.15 SD =.74 3 (6.2%) M =.19 SD = (2.6%) Max = (2.1%) Max = (.07%) Max = (1.3%) Max = (2.6%) Max = 15

43 Table 3: continued 43 Hiding relationship from others Watching what you say around heterosexuals People staring because you are LGBT Verbal harassment Treated unfairly stores/restaura nts People laughing making jokes Hearing about people you know treated unfairly Hearing about crimes towards unknown Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 7 (13.2%) M =.40 SD = (50.9%) M = 1.19 SD = (13.2%) M =.28 SD =.91 1 (1.9%) M =.02 SD =.14 1 (1.9%) M =.02 SD =.14 0 M = 0 SD = 0 20 (37.7%) M =.57 SD = (43.4%) M =.92 SD = (13.5%) M =.29 SD = (40.3%) M =.94 SD = (11.5%) M =.19 SD =.63 0 M = 0 SD = 0 0 M =0 SD = 0 2 (3.8%) M =.19 SD = (21.6%) M =.37 SD = (28.8%) M =.58 SD = (9.8%) M =.37 SD = (21.6%) M =.61 SD = (6%) M =.10 SD =.46 1 (1.7%) M=.02 SD =.14 0 M = 0 SD = 0 0 M = 0 SD = 0 7 (13.7%) M =.29 SD = (29.4%) M =.61 SD = (19.6%) M =.37 SD = (30.4%) M =.57 SD = (20%) M=.20 SD =.40 0 M = 0 SD = 0 2 (4.3%) M =.04 SD =.21 4 (8.7%) M =.13 SD = (21.3%) M =.24 SD =.67 8 (17.4%) M =.52 SD = (10.6%) M =.21 SD = (30.4%) M=.81 SD = (8.5%) M=.17 SD =.64 2 (4.3%) M =.04 SD =.20 1 (2.2%) M =.02 SD =.14 0 M = 0 SD = 0 10 (21.3%) M =.30 SD =.69 5 (10.6%) M =.19 SD =.65 5 (10.6%) M =.19 SD = (31.9%) M =.62 SD = (4.2%) M=.09 SD =.46 2 (4.3%) M =.04 SD =.21 2 (4.2%) M =.04 SD =.21 1 (2.1%) M =.02 SD = (21.3%) M=.30 SD =.57 6 (12.8%) M =.21 SD =.66 5 (10.6%) M =.26 SD = (22.9%) M=.48 SD = (8.3%) M=.17 SD =.63 0 M = 0 SD = 0 0 M = 0 SD = 0 4 (4.2%) M =.04 SD = (20.8%) M =.29 SD =.68 9 (18.7%) M =.31 SD =.78 Daily Sum Averaged Across Week (3.6%) Max = (9.1%) Max = (1.5%) Max = (.21%) Max = 2.86 (.21%) Max = (.67%) Max = (4%) Max = (5.9%) M in = 0 Max = 49

44 Table 3: continued 44 Hearing others being called names Hearing about unfair treatment of unknown Hard finding LGBT friends Few people to talk to about being LGBT Don t fit in with other LGBT Difficulty finding partner Grand Total Averaged Across Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 11 (20.7%) M =.40 SD = (43.4%) M =.92 SD = (41.5%) M =.91 SD = (45.2%) M = 1.21 SD = (39.6%) M =.77 SD = (35.8%) M = (11.5%) M =.15 SD = (23.1%) M =.46 SD = (28.8%) M =.71 SD = (36.5%) M =.90 SD = (21.1%) M =.37 SD =.93 16(30.8%) M =.81 4 (7.8%) M =.12 SD = (21.6%) M =.49 SD = (23.5%) M =.59 SD = (27.4%) M =.71 SD = (19.6%) M =.35 SD =.84 9 (17.6%) M =.57 5 (10.9%) M =.15 SD = (30.4%) M =.61 SD = (28.3%) M =.67 SD = (28.2%) M =.59 SD = (23.9%) M =.41 SD = (23.9%) M =.59 SD = 2.20 SD = 1.61 SD = 1.45 SD = (2.2%) (1.5%) (1.2%) (1.2%) Max = 65 Max = 49 Max = 36 Max = 31 Stressors 3 Note. M = mean times stressor was endorsed by individual, range Number of individuals endorsing any occurrence of the minority stressor by day 2. Total daily sum averaged across the week, across all individuals 3. Grand total averaged across all stressors, across all individuals 4 (8.5%) M =.13 SD = (21.3%) M =.34 SD = (23.4%) M =.53 SD = (25.5%) M =.72 SD = (12.8%) M =.30 SD =.95 9 (19.1%) M =.57 SD = (1.1%) Max = 38 2 (4.2%) M =.09 SD =.46 9 (19.6%) M =.43 SD = (21.3%) M =.43 SD = (25.5%) M =.47 SD =.95 8 (17%) M =.55 SD = (23.9%) M =.49 SD = (.9%) Max = 29 6 (12.5%) M =.17 SD = (27.1%) M =.56 SD = (12.8%) M =.30 SD = (29.2%) M =.50 SD = (16.7%) M =.29 SD =.77 9 (18.7%) M =.58 SD = (.9%) Max = 28 Daily Sum Averaged Across Week (4%) Max = (6.6%) Max = (7.2%) Max = (8.9%) Max = (5.3%) Max = (8.5%) Max = (1.3%) Max = 39.42

45 Diurnal Cortisol 45 As seen in Figure 2, diurnal cortisol across participants pattern best fit a cubic trajectory of time F(1, 316) = 15.88, p < The fixed cubic effect of time suggests that cortisol levels increased from wake to 45 minutes, decreased over the subsequent 12 hours, and continued to decrease at a slower rate up until bedtime. Inter-class correlations (ICC) for total cortisol output (AUCg), or the proportion of variance in AUCg that can be explained within and between individuals, revealed that 34% of the variability in AUCg can be attributed to within-person differences and 66% of the variability in AUCg can be attributed in between-person differences.

46 46 Salivary Cortisol (nmol/l) Figure 2. Average diurnal cortisol slopes (n=48) Time since wake (hours)

47 Daily Minority Stress and Diurnal Cortisol 47 The first aim of the current study was to examine the association between daily minority stressors and daily cortisol output over the span of a week in a sample of sexual minority young adults. The following were included as covariates: Outness, time of disclosure, typical alcohol and tobacco use, typical physical activity, and wake time. It was hypothesized greater self-reported sexual minority stress would be associated with greater cortisol output across the week. Using the first daily minority stress coding scheme, it was found that average daily minority stress was not associated with diurnal cortisol output across the week, F(1, 148) = 1.67, p =.20. The second coding scheme also resulted in a null finding regarding minority stress and diurnal cortisol output, F(1, 148) = 1.35, p =.25. The third coding scheme resulted in a null finding as well, F(1, 42) = 2.00, p =.16. None of the minority stress subscales were associated with diurnal cortisol either, F s < 2.00, p s >.17. As an exploratory aim, diurnal cortisol slope was also examined with regards to daily minority stress, however none of the minority stress coding schemes were associated with linear or cubic diurnal cortisol slope, F s < 1.45, p s >.25. Hardiness and Diurnal Cortisol The second aim of the current proposed study was to examine psychological hardiness as a potential source of resiliency in sexual minority individuals. It was hypothesized that: (a) there would be a direct association between hardiness and cortisol output, such that greater hardiness would be associated with lower cortisol output across the week (Hypothesis 2a). Results indicated that hardiness did not predict total cortisol output across the week, F (1,54) =.51, p =.45. As an exploratory analysis, the

48 48 association between hardiness and cortisol slope was also examined. It was hypothesized that greater hardiness would be associated with steeper cortisol slopes. Results indicated that hardiness was not related to linear, F (1,742) = 1.42, p =.22, or cubic, F (1,742) = 1.36, p =.24, diurnal cortisol slope. It was further hypothesized that (b) there would be an interaction between hardiness and daily sexual minority stressors (averaged across the week) and cortisol output across the week, such that greater hardiness would weaken the association between daily stressors averaged across the week and cortisol output across the week (Hypothesis 2b). There was not a significant interaction between hardiness and sexual minority stressors across the week using any of the three coding schemes, F s < 2.00, p s >.20. There was also not a significant interaction between hardiness and sexual minority stressors on cortisol linear or cubic slope, F s < 1.00, p s >.70 (Hypothesis 4). In order to further examine the association between hardiness and diurnal cortisol, the associations between the three subcomponents of hardiness (control, challenge, and commitment) and total cortisol output were examined. Again, the control subcomponent refers to having a sense of control over the events of one s life. The challenge subcomponent refers to seeing obstacles or problems as challenges to overcome instead of threats. Finally, the commitment subcomponent refers to a genuine commitment to actively engage in life despite setbacks. Results indicated that none of the subcomponents were associated with total cortisol output across the week F s <.60 p s >.44. Diurnal cortisol slope was also examined in relationship with the hardiness subscales. Results indicated that the challenge subcomponent was associated with cubic

49 49 diurnal cortisol slope across the week, b =.0004, SE =.0001, F (1, 741) = 5.64, p =.01. As seen in Figure 3, those more likely to see stressors as obstacles rather than challenges to overcome (low challenge) did not have the prototypical rise between wake time and 45 minutes after wake time. In contrast, those more likely to see stressors as challenges to overcome (high challenge) had a more robust rise in cortisol at wake, F (1,498) = 4.78, p =.03, and a steeper decline in cortisol 12 hours post wake, F (1,498) = 4.44, p =.04. The potential interaction between the challenge subcomponent of hardiness and minority stress across the week was also examined. None of the coding schemes of minority stress significantly interacted with the challenge subcomponent to predict linear or cubic diurnal cortisol slope across the week F s < 1.00, p s >.75. In other words, the nature of the association between minority stress and cortisol slope did not differ as a function of challenge scores.

50 Figure 3. Temporal changes in raw salivary cortisol levels as predicted the challenge subcomponent of psychological hardiness. High (low) levels correspond to one standard deviation above (below) mean levels. 50

51 Additional Exploratory Analyses 51 To further examine the association between daily stress and diurnal cortisol in sexual minority young adults, the association between general negative events (averaged across the week) and diurnal cortisol was examined. As can be seen in Table 4, general negative events were endorsed at slightly higher rates compared to daily minority stressors (the total number of negative events across the week for the average person was 35). For example, unlike minority stressors, every negative event was endorsed at least once throughout the seven-day testing period. Some of the most commonly endorsed negative events included: not having enough time to meet obligations (12%), cash flow difficulties (9%), and social isolation (7%). The least endorsed negative events included conflict with family or friends (3%) and being disappointed by friends (2%). Results of analyses testing the relationship between general negative daily events and cortisol output indicated that greater general negative events across the week was associated with greater diurnal cortisol output (AUCg), b =.07, SE =.02, F (1, 44) =13.36, p = See Figure 4 for the predicted total cortisol output (averaged across the week) as a function of self-reported negative events (averaged across the week) at one standard deviation above and below the mean. As shown, more negative events was related to greater daily cortisol output. Daily negative events were not associated with either linear F (1,710) =.02, p =.87 or cubic F (1, 710) =.03, p =.85, diurnal cortisol slope. Neither hardiness nor the challenge subcomponent moderated the association between cortisol output or cortisol slope, F s < 1.00 p s >.60.

52 Table 4 52 Endorsement of negative event by day for survey plus cortisol arm of study (N = 58) Conflict with friends/family Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 8 (15%) M =.31 SD = (19%) M =.38 SD = (16%) M =.31 SD = (17%) M =.55 SD =.31 3 (6%) M =.22 SD =.55 5 (11%) M =.11 SD =.48 4 (8%) M =.15 SD =.51 Daily Sum Averaged Across Week (3%) Max = 20 Disappointed by friends 11(21%) M =.25 SD =.55 9 (17%) M =.29 SD =.85 8 (16%) M =.27 SD =.85 3 (6%) M =.07 SD =.25 3 (6%) M =.06 SD =.25 8 (17%) M =.26 SD =.68 6 (12%) M =.25 SD = (2%) Max = 15 Dissatisfied with work 25 (47%) M =.96 SD = 1.4 Disliking work 30 (59%) M =.96 SD = 1.44 Too many things to do at once No time to meet obligations 34 (64%) M = 1.66 SD = (51%) M = 1.38 SD = (43%) M =.98 SD = (36%) M = 1.02 SD = (50%) M = 1.62 SD = (48%) M = 1.48 SD = (25%) M =.61 SD = (27%) M =.67 SD = (41%) M = 1.27 SD = (35%) M = 1.10 SD = (31%) M =.97 SD = (28%) M =.67 SD = (35%) M =.78 SD = (31%) M =.78 SD = (25%) M =.53 SD = (28%) M =.66 SD = (28%) M =.79 SD = (23%) M =.70 SD = (25%) M =.64 SD = (30%) M =.51 SD = (30%) M =.72 SD = (32%) M =.62 SD = (19%) M =.52 SD= (25%) M =.60 SD = (37%) M =.67 SD = (37%) M =.81 SD = (8%) Max = (9%) Max = (13%) Max = (12%) Max = 77 Cash flow difficulties 21 (40%) M = 1.08 SD = (31%) M =.69 SD = (31%) M=.78 SD = (28%) M =.67 SD= (23%) M =.66 SD = (28%) M =.79 SD = (21%) M =.50 SD = (9%) Max = 57

53 Table 4: continued 53 Financial burdens Social isolation Taken advantage of Grand Total Averaged Across Stressors 3 Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 27 (51%) M =1.22 SD = (43%) M = 1.02 SD = (38%) M = 1.22 SD = (9%) Max = (36%) M =.94 SD = (27%) M =.69 SD = (26%) M =.80 SD = (8%) Max = (36%) M =.88 SD = (33%) M =.65 SD = (27%) M =.82 SD = (6%) Max = (33%) M =.80 SD = (22%) M =.31 SD = (22%) M =.67 SD = (4%) Max = (28%) M =.66 SD = (11%) M =.15 SD =.51 8 (15%) M =.50 SD = (4%) Max = (30%) M =.83 SD = (23%) M =.49 SD = (9%) M =.20 SD = (4%) Max = 39 9 (19%) M =.52 SD = (21%) M =.50 SD = (9%) M =.21 SD = (4%) Max = 39 Daily Sum Averaged Across Week (10%) Max = (7%) Max = (4%) Max = (6%) Max = 55.6 Note. M = mean times an individual endorsed negative events each day, range = Number of individuals endorsing any occurrence of the event by day 2. Total daily sum averaged across the week, across all individuals 3. Grand total averaged across all stressors, across all individuals

54 Figure 4. Total cortisol output (averaged across the week) predicted by self-reported negative events (averaged across the week) at one standard deviation above and below the mean. 54

55 The association between daily positive events and diurnal cortisol in sexual 55 minority young adults was also tested. As seen in Table 5, daily positive events were endorsed at a much greater rate than daily negative events or daily minority stressors (the total number of positive events across the week for the average person was 83). Daily positive events were coding using the same coding scheme for daily negative events. The most commonly endorsed positive events were: good interactions with friends (31%), laughed a lot with family and friends (31%) and sent a pleasant or message (32%). Some of the least endorsed positive events were did something special that was appreciated (12%) and caught up or got ahead in work (7%). As shown in Figure 5, results indicated that greater daily positive events were associated with lower cortisol output, b = -.009, SE =.002, F(1,44) =.17.14, p =.001. Positive events were also associated with cubic diurnal cortisol slope, b = , SE =.00003, F(1,730) = 5.34, p =.02. As seen in Figure 6, participants who reported less positive events (averaged across the week) had higher cortisol levels at wake and at 45 minutes after wake time compared to participants who reported more positive events (averaged across the week), F(1, 491) = 4.97, p = 03, but these difference seem to diminish at 12 hours after wake time F(1, 491) = 2.00, p =.16, and at bedtime F(1,491) =.39, p =.53.

56 Table 5 56 Endorsement of positive event by day for survey plus cortisol arm of study (N = 58) Went socializing with friends/date Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 23 (55%) M =.96 SD= (50%) M =1.00 SD = (49%) M = 1.24 SD = (65%) M = 1.83 SD = (74%) M = 2.19 SD = (57%) M = 1.60 SD = (60%) M = 1.60 SD = 1.81 Daily Sum Averaged Across Week (18%) Max = 101 Good interactions with friends Progress made towards task with deadline Pleasant time with friends/family Compliment from partner/friend/fa mily Did something special that was appreciated Did something special that was appreciated 42 (79%) M = 2.38 SD = (64%) M = 2.60 SD = (75%) M = 1.55 SD = (60%) M = 1.58 SD = (49%) M =.79 SD = (49%) M =.79 SD = (88%) M = 3.00 SD = (63%) M = 1.90 SD = (73%) M = 1.96 SD = (54%) M = 1.08 SD = (33%) M =.67 SD = (33%) M =.67 SD = (80 %) M = 2.37 SD = (53%) M = 1.67 SD = (80%) M = 2.14 SD = (59%) M = 1.69 SD = (59%) M = 1.06 SD = (59%) M = 1.06 SD = (83%) M =2.39 SD = (56%) M = 1.30 SD = (72%) M = 2.37 SD = (56%) M = 1.74 SD = (43%) M = 1.35 SD = (43%) M = 1.35 SD = (83%) M =3.17 SD = (53%) M = 1.51 SD = (83%) M = 2.74 SD = (55%) M = 1.57 SD = (49%) M = 1.34 SD = (49%) M = 1.34 SD = (74%) M = 2.23 SD = (53%) M=.1.47 SD = (76%) M = 2.30 SD = (53%) M = 1.43 SD = (40%) M = 1.04 SD = (40%) M = 1.04 SD = (83%) M =2.29 SD = (56%) M =1.56 SD = (75%) M = 2.21 SD = (64%) M = 1.35 SD = (47%) M =.94 SD = (47%) M =.94 SD = (31%) Max = (20%) Max = (26%) Max = (18%) Max = (12%) Max = (12%) Max = 63

57 Table 5: continued 57 I laughed a lot with friends/ family Day 1 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 38 (72%) M = 2.52 SD = (71%) M = 2.48 SD = (67%) M = 2.39 SD = (76%) M = 2.80 SD = (81%) M = 2.85 SD = (70%) M = 2.40 SD = (77%) M = 2.52 SD = 2.12 Daily Sum Averaged Across Week (31%) Max = 134 Completed work on interesting project Caught up (got ahead) Pleasant /message sent Grand Total Averaged Across 23 (43%) M = 1.26 SD = (55%) M = 1.57 SD = (72%) M = (54%) M = 1.15 SD = (38%) M = 1.12 SD = (71%) M = (43%) M= 1.18 SD = (39%) M = 1.29 SD = (67%) M = (33%) M=.76 SD = (39%) M =.96 SD = (76%) M = 2.95 SD = 2.53 SD = 2.40 SD = 2.43 SD = (15%) 83 (14%) (15%) (14%) Max = 134 Max = 129 Max = 122 Max = 129 Stressors 3 Note. M = mean times an individual endorsed positive events each day, range = Number of individuals endorsing any occurrence of the event by day 2. Total daily sum averaged across the week, across all individuals 3. Grand total averaged across all stressors, across all individuals 13 (28%) M=.74 SD = (33%) M =.98 SD = (81%) M = 3.00 SD = (15%) Max = (47%) M = 1.32 SD = (45%) M = 1.09 SD = (70%) M = 2.40 SD = (13%) Max = (42%) M =.96 SD = (30%) M =.94 SD = (77%) M = 2.50 SD = (13%) Max = (13%) Max = (7%) Max = (32%) Max = (14%) Max = 126

58 Figure 5. Total cortisol output (averaged across the week) predicted by self-reported positive events (averaged across the week) at one standard deviation above and below the mean. 58

59 Figure 6. Temporal changes in raw salivary cortisol levels as predicted by self-reported positive events averaged across the week. High (low) levels correspond to one standard deviation above (below) mean levels of positive events. 59

60 Discussion 60 Sexual minorities are a high risk group whose experience of daily stress, and its subsequent impact on health, has been under examined. The current study aimed to further examine daily stress processes in sexual minority young adults and their association with an essential health-relevant biomarker, diurnal cortisol. Further, factors that confer resiliency are under studied in sexual minorities as well. In this study the association between psychological hardiness, a potential resilience factor, and diurnal cortisol was also examined. This first aim of the current study was to examine the association between minority stress, averaged across the week, and cortisol output. Results indicated that averaged daily minority stress was not associated with cortisol output (or cortisol slope), contrary to expectations. As previously stated, past research has shown that recent major minority stress may be associated with flatter diurnal cortisol slopes in a sexual minority population (e.g., Parra et al., 2016). It should be noted that the current study was the first to examine daily minority stressors and diurnal cortisol. One reason that might account for this result is the general lack of endorsement of minority stressors throughout the week. Several alternative coding schemes were examined due to the lack of endorsement and none of them showed an association with diurnal cortisol. Previous studies examining the association between daily minority stressors and self-reported mental health used open ended diaries that were then coded by independent coders for evidence of minority stress (e.g., Swim et al., 2007). However, as in the current study were averaged endorsed minority stressors was about 15, those studies also endorsed few minority stressors across the week (M = 2). The current study

61 61 also allowed participants to write in any additional stressors that occurred during the day, but none endorsed stressors specific to their sexual minority status. One reason for this may be that the correct questions were not being asked. Perhaps there should have been more questions regarding vicarious discrimination or isolation since these seemed to be the most endorsed in the current sample. Alternatively, one could argue that the lack of endorsement of minority stressors may be indicative of general societal change (i.e., either discrimination is truly declining or it is perhaps not as overt). In the summer of 2015, while data collection was taking place, nationwide marriage equality was made law by the Supreme Court of the United States. However, a year later, towards the end of data collection, the largest mass shooting in U. S. history occurred at the gay nightclub, Pulse, in Orlando, Florida. Given the current political climate, it will be increasingly important to continue to study daily stressors in sexual minorities to observe whether these processes and their potential impacts change over time. Finally, it could be the case that the null finding in the association between minority stress and diurnal cortisol, in the current sample, may be correct. The current sample reported very high levels of social support (M s = on a scale from 5 to 16) and higher levels of daily positive interactions with family and friends. Given the snowballing recruitment strategy of the current sample, it could be the case that these individuals had large social networks from which to draw from, thus increasing the number of participants with greater levels of social support. As previous research has shown (Cohen, 2004), social support is positively associated with well-being and health. Although not related to cortisol in the current study, social support may still be a source

62 of resilience in the current sample of sexual minority young adults. Further, it could 62 simply be the case that the areas from which we sampled from have policies in place that protect sexual minorities and therefore make discrimination less likely to occur. It should be noted that the current study was powered to detect medium to large effect sizes. It could be that the association between daily minority stressors and diurnal cortisol is a small one, and therefore, the current study may have been underpowered. The second aim of the current study was to examine the role of psychological hardiness as a potential source of resilience. Psychological hardiness was examined in the current study due to its past association with both direct and stress buffering effects on self-reported health in sexual minorities (Figueroa & Zoccola, 2015). In the current study, psychological hardiness was negatively associated with alcohol and tobacco use across the week, depressed mood, anxiety and general stress and positively associated with social support and engagement in physical activity (see Table 1). Although the hardiness scale total score was not associated with cortisol output or slope, the subcomponent challenge was significantly associated with cortisol slope. Specifically, there was a lack of the prototypical rise in cortisol 45 minutes after wake and a lack of decline in cortisol at bedtime in those more likely to see negative events as stressors rather than challenges to overcome. As previously stated, non-normal diurnal cortisol patterns may be indicative of dysregulation which in the long term may lead to disease (Chrousos & Gold, 1992; McEwen, 1998). It is important that society encourage the reduction of discriminatory behavior; until that happens it may be useful to provide individuals with coping skills that may be helpful for dealing with minority stress and other stressors. If seeing stressors as challenges to overcome instead of obstacles is

63 associated with better health (i.e., steeper cortisol slopes in the current study) perhaps 63 interventions designed to train individuals to reframe stressors as challenges to overcome would be beneficial. Hardiness training is currently used in many college campuses (Khoshaba & Maddi, 2001; Maddi et al., 2009), and previous research has shown that hardiness training not only significantly increases hardiness, but is also beneficial for atrisk undergraduates with regards to retention rates and grade point average (Maddi et al., 2002). Similar kind of trainings could potentially be offered at LGBT centers at universities, particularly in the Midwest or small-town and rural locations where often times these student centers are the only source for the LGBT community (Swank, Frost, & Fahs, 2012). Although hardiness did not moderate the association between minority stress (or negative events) and diurnal cortisol in the current study, hardiness was still associated with better mental health variables (e.g., less depressed mood) and negatively associated with risky health behaviors (e.g., smoking). Future studies should further continue to examine both direct and indirect effects hardiness may have diurnal cortisol as well as other physiological markers of health (e.g. cardiovascular). Additional exploratory analyses examined daily negative and positive events, averaged across the week, and their association with diurnal cortisol. Although the main aim of the current study was to examine stressors unique to sexual minorities, it is important to examine general daily stressors as they may be detrimental to health over time (for review, see Almeida, 2005). Results indicated that greater negative events, averaged across the week, was associated with greater cortisol output over the week. Again, higher cortisol output has been associated with poor health outcomes (e.g., cardiovascular disease; Chrousos, 2009; Kirschbaum et al., 1995). These findings extend

64 64 previous cross sectional work on the positive association between general stress and poor self-reported health in sexual minorities (e.g., Frost et al., 2013). Further, these findings are in line with other national samples examining the general population in which daily stressors were also found to be associated with higher cortisol output (Stawski et al., 2013). With regards to positive events, it was found that greater positive events across the week was associated with less cortisol output and steeper cortisol slopes across the week (i.e., a healthier cortisol response). These findings extend previous cross sectional findings with regards to sexual minority young adults positive experiences with family and friends and its association with better self-reported health (e.g., less depressive mood) and decreased suicidality (Ryan et al., 2010). Further, although the association between daily positive events and cortisol has not been previously examined, the current results do support other studies that have examine other high risk groups that have found similar results (i.e., greater positive events were associated with less cortisol output in women with risky pregnancies; Pleuss et al., 2012). Also, positive daily events have been related to lower inflammation in a recent national study (Sin et al., 2015). In sum, although the original intent of the current study was to examine the association between minority stress and diurnal cortisol, given the association between daily events and health in general population, it is important to know how daily general events (both positive and negative) may influence health outcomes in sexual minorities. Current findings suggest that experiencing daily uplifts or daily positive events may also confer some biological resilience.

65 65 Some strengths of the current study include examining daily events in a weeklong diary design as well as assessing an objective marker of stress, cortisol. The inclusion of the biomarker cortisol, via ecological momentary assessment of saliva, represents a novel and multilevel approach to examining sexual minority health, stress and well-being. Assessment of diurnal salivary cortisol allows for the linking of dynamic psychosocial stress processes to an important neuroendocrine indicator of stress, which in turn, may be predictive of disease progression and mortality. Also, the use of electronic daily diaries allowed the researchers to capture daily variations in the stress processes while minimizing concerns of confidentiality. Further, unlike previous studies examining sexual minorities and cortisol (i.e., Burton et al., 2014; Hatzenbuehler & McLaughlin, 2013; Parra et al., 2016) that have focused on large urban metropolitan areas, the current study focused on the Midwest (45.5% from Ohio), a largely understudied population within the sexual minority community. This is particularly important given the rate at which isolation was reported in the current study (both in the sexual minority specific events and the general negative events). Although research on sexual minority health in urban areas provides important information, it is largely unknown the extent to which such findings apply to sexual minorities in rural and small towns, including in the Midwest, where LGBT resources may be fewer and stigma greater (Swank, Frost, & Fahs, 2012). Gathering information on daily processes and health in this especially vulnerable population will be essential to further understanding the mechanisms linking stress and health in sexual minorities. Some limitations of the current study include the time-limited nature of the study and the narrow age range of the sample. Although data was collected throughout the year,

66 66 it could be the case that in the week we assessed the participant they did not experience as many stressors as they did in subsequent or prior weeks. It will be necessary to do longitudinal designs in order to follow up with the findings of the current study. Further the age of the sample was restricted to individuals 18 to 35 in order to control for covariates associated with diurnal cortisol (e.g., older individuals are more likely to have endocrine or major chronic disorders that may influence cortisol). Sexual minority youth and sexual minority older adults may experience daily stressors at different rates or different stressors altogether. In order to better understand daily stress processes and their association with health, future research will need to assess sexual minorities across the lifespan. Lastly, the sample was not very diverse with regards to race and ethnicity and there were also no transgender individuals in the final sample. It will be important to examine a more diverse sample of sexual minorities to more fully understand the association between daily minority stress and diurnal cortisol. However, the current sample of sexual minority adults was comparable to other national samples of sexual minorities with regards to lifetime discrimination, stigma consciousness, general stress, depressed mood, anxiety and psychological hardiness (e.g., Figueroa & Zoccola, 2016). In conclusion, the current study was the first to examine daily stress processes and their associations with diurnal cortisol in a sample of sexual minority young adults. Further, the study was also the first to examine psychological hardiness as its association with diurnal cortisol in sexual minorities. Findings from the current study suggest that seeing stressors and challenges to overcome and experiencing daily positive events were related to lower cortisol output and steeper cortisol slopes. Negative daily events (but not minority-specific events) predicted greater diurnal cortisol output. Continued research on

67 the association between daily processes and diurnal cortisol in sexual minorities is 67 needed. Additionally, as the current study suggests, further exploration on how the subcomponents of psychological hardiness may be associated with biological processes in sexual minorities is warranted.

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