Biparental care has evolved to ensure successful rearing of offspring. However, separation during periods of care can lead to conflicts that might negatively impact pair bonds and offspring care. In this study, pair-bonded convict cichlids (Amatitlania nigrofasciata) were observed for changes in behavior toward their partners and offspring before and after a period of separation. Males and females were designated either as a Resident (remain with offspring) or Removed (separated from partner and offspring for 5 days) individual. Behaviors between partners and toward offspring were measured before and after separation, and compared to the levels of behavior of control pairs (never separated), as well as individuals introduced to a novel partner instead. Cortisol levels of Resident male and female A. nigrofasciata were assayed using water-borne hormone collection before and after separation. Aggression between pair bond members did increase following reintroduction, but did not lead to the termination of pair bonds. Resident females showed more aggression to novel partners than Resident males. Offspring care decreased in both Resident and Removed females. Experimental pairs decreased the amount of time spent interacting with intruders. Cortisol levels were significantly higher among experimental pairs compared with control pairs that did not experience a separation. Females (both control and experimental) showed small, yet significant increases in cortisol levels, while both control and experimental males did not. These results suggest that while pair bonds appear resilient, prolonged separations influence pair bond and parental care dynamics, both behaviorally and hormonally, and require pairs to re-establish roles, resulting in less time caring for offspring.