This study examined salivary cortisol levels in couples in which one member had unexplained chronic fatigue (CF). The couples completed questionnaires and seven household activities in a laboratory setting and provided salivary cortisol samples prior to and immediately after the activities, as well as again after completing additional questionnaires and debriefing. The couples rated their interactions as similar to those at home, suggesting ecological validity, and patients with CF experienced the activities as involving more exertion than did their partners. The multilevel model results indicated that patients with CF had overall lower cortisol levels and flatter slopes across repeated measurements than did their significant others. Patients’ and significant others’ cortisol concentrations were significantly associated with each other over time. Furthermore, significant others’ cortisol was associated with greater relationship satisfaction and greater observed rates of patients’ illness/pain behaviors per minute, but patients’ levels of cortisol were not associated with relationship variables. This study is the first to examine cortisol in couples with CF; the results are discussed in terms of implications for future research.