This study investigated changes in day-to-day affect, behavior, and physiology (hypothalamic-pituitary-adrenocortical axis activity) associated with temporary physical separations from romantic partners (such as those brought about by work-related travel). Daily diaries and measures of salivary cortisol were collected from 42 couples over a 21-day period that was timed to coincide with a naturally occurring 4- to 7-day separation. There were significant changes from preseparation to separation and from separation to reunion in the quality of partners' interactions, their positive and negative affect, sleeping problems, subjective stress, physical symptoms, and cortisol levels. Separation and reunion effects were generally more pronounced in homebound partners, partners with high attachment anxiety, and partners who had less contact with each other during the separation. Separation and reunion effects were not moderated by relationship length, relationship satisfaction, how often couples underwent separations, or the presence of children in the home. The results are discussed with respect to the role of daily proximity and contact between partners for day-to-day affective and physiological regulation.