This study examined job fatigue and stress reactions in 75 male Japanese ambulance team members of the X City Fire Bureau, using the job fatigue questionnaire of “Jikaku-sho shirabe”. Results showed that perceptions of both drowsiness and eyestrain were significantly higher at the end of 24-shift than at the start in members who were dispatched more than 7 times (both p<0.01), and that the longer the hours of both emergency dispatch duty and dispatch-related duty, the higher the significant scores of both drowsiness and eyestrain. Using salivary amylase activity (sAMY) as a stress reaction, results showed that the mean values of sAMY of ambulance team members were higher than those of Japanese male young adults, suggesting that Japanese ambulance team members had high levels of job stress. We predicted that sAMY values at the end of 24-shift would show an increase compared to those at the start, indicating the influence of workload. However, our results were contrary to this prediction. We also observed no correlation between variation in sAMY and work conditions, or between variation in sAMY and variation in levels of job fatigue. In order to assess the health of urban ambulance team members using sAMY, measurements must be made before the end of duty shifts, while members' workload and stress are still ongoing.