Reward and punishment play an important role in clinical education. A series of studies have indicated that the clinical effect of reward and punishment can be affected by the frequency, individual sensitivity and the expectation of reward and punishment. However, little is known about the timeliness of the role of reward and punishment. In the present study, we investigated the length of time for which the emotion triggered by cumulative reward/punishment could act on inhibition ability. This was investigated using a psycho-physiological methodology which used the stop-signal task to explore the timeliness of reward and punishment. The experiment comprised a 3 (group: reward group, punishment group, and control group) × 5 (periodicity: stage 1, stage 2, stage 3, stage 4, and stage 5) mixed factorial design, in which stimulus condition varied between subjects, and periodicity varied within subjects. Forty-five college students were allocated to the reward group, punishment group, or control group at random. The experimental process was divided into five latent stages. The experiment used the Super Lab system to present the stimuli, and to record the response time and rate of error inhibition made by participants in the stop-signal task during each stage. Automatic physiological responses were collected continuously by a 16-channel physiological recording system during each stage. The results showed that (1) differences in behavioral response time and error inhibition rate only occurred in Stages 2 and 3. Specifically, the response time of the reward group in Stage 2 was much higher than that in the control group; the response time of the reward group in Stage 3 was much higher than those observed in the punishment or control group; and the error inhibition rate of the reward group in Stages 2 and 3 was much lower than in the punishment or control group; (2) For automatic physiological responses, the impact of reward/punishment stimulations differed in almost all stages for skin conductance responses and finger temperature, but not heart rate. In conclusion, the present study demonstrated that the effects of reward/punishment stimulations on behavioral inhibition ability differed across time. At the critical time periods, only the reward condition improved behavioral inhibition ability; and the punishment condition increased the inhibition response. The effects of reward stimulations across time differed for heart rate, but not skin conductance responses or finger temperature activities.