It was hypothesized that an individual's chronotype might influence the response to physical activity at a given time of day. This study aimed to analyze the psychophysiological responses during a walking task at different times of day in individuals with different chronotypes. 46 students ( M age = 24.8yr., SD = 7.2) filled in the Morningness-Eveningness Questionnaire to determine chronotypes. Heart rate, walking time, and the rating of perceived exertion (RPE) were measured during two self-paced walking sessions: one in the morning (08:30) and one in the afternoon (15:30). A multivariate analysis of variance found a significant interaction between chronotype and time of day. The post hoc analysis showed a significant difference for RPE in the morning session, with evening types reporing a higher RPE compared with the morning types. The chronotype and the time of day when a physical task is undertaken can influence the RPE response, although it might not influence physiological or performance parameters. This has to be taken into account, because it can affect test reliability as well as possibly have a negative influence on the affective responses to a given task.