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Objective There is limited knowledge about how fatigue develops and worsens and what influences fluctuations in daily fatigue. Stress was found to influence fatigue, and being in a relationship seems to either increase or decrease stress depending on the couple interaction. In this study, co-variation of fatigue, self-reported stress, and biological stress markers in couples’ everyday lives was investigated. Specifically, we examined a) whether momentary couple interactions moderated dyadic outcomes and b) whether and how stress and relationship measures influenced individual momentary fatigue. Methods Forty heterosexual couples (age: 28 ± 5 years) reported subjective fatigue and stress levels 4 times a day for 5 consecutive days (1600 measures). Furthermore, participants reported whether they had interacted with their partner since the last data entry and, if so, they rated the valence of this interaction. Salivary cortisol (a measure of HPA axis activity) and alpha amylase (a measure of ANS activity) were analyzed as biological stress markers from saliva samples obtained at the same time points. Moment-to-moment data were analyzed using dyadic multilevel models to account for the nested design. Results Stress (women and men: p ≤ 0.001) and fatigue (women: p = .003, men: p = .020) showed patterns of co-variation within couples, especially if partners had interacted with each other since the previous data entry. Cortisol was also found to co-vary between partners (women: unstandardized coefficient (UC) = 0.12, p ≤ .001, men: UC = 0.18, p ≤ .001), whereas the regulation of alpha-amylase levels depending on the partner’s levels was only present in women (UC = 0.11, p = .002). Valence of couple interaction was negatively associated with fatigue (women: UC = −0.13, p ≤ .001, men: UC = −0.06, p = .011). There was no momentary association of fatigue with an individual’s own or the partner’s subjective or biological stress markers. Conclusions Fatigue and stress levels during the day seem to co-vary within couples. These associations were particularly strong when the partners had interacted with each other since the last measurement. These data underline the importance of social factors in fatigue and stress in everyday life.