This study examined the relationship between trait emotional intelligence (EI) and variation in psychological (positive affect: PA, negative affect: NA) and psychophysiological (salivary alpha‐amylase: sAA) indicators among Japanese employees over 3 consecutive days (working day 1, non‐working day, working day 2). The analyses revealed that higher trait EI was associated across the days with higher PA, but not with NA. Moreover, diurnal sAA levels were lower in the high trait EI group than in the low trait EI group on the intervening non‐working day, and this difference between the EI groups continued to show a tendency to significance on working day 2. The results indicate that higher EI may be related to the preservation of higher levels of PA and lower levels of sympathetic activity in recovery in the naturalistic condition.