Guinea pigs (Cavia porcellus) are used for interactive activities in zoos; therefore, it is important to investigate their welfare. This research aimed to evaluate the validity of measuring the guinea pigs’ body temperature of guinea pigs through the ear canal and investigate the relationship among changes in the expression of negative behavior, changes in body temperature, and changes in salivary cortisol concentration, and examine the effects of different interactive activities. In the normal interactive activities performed at the site, the decreased body temperature of pigs was observed over time. In contrast, increased body temperature was observed in excessive interactive activities, which are not recommended. Among the negative behaviors, “Head turning” and “Locomotion” increased significantly in excessive interactions compared to normal interactions, but “Head tossing” decreased significantly over time in both types of interactions. “Freezing” was observed only in excessive interactions. Salivary cortisol concentration increased significantly for all activities. Investigating the relationship between the individual expression of negative behavior and changes in body temperature and changes cortisol level made it possible to uncover the potential for inferring an animal’s physiological state. Combining ear temperature monitoring and behavioral observation during zoo interaction activities is recommended as an ethical and scientifically supported practice.