Manual handling of chickens is required for many veterinary, research, and breeding procedures. This study aimed to assess the changes in physiological parameters over time during manual restraint of chickens, as well as the effect of hooding on these parameters. Heart rate, heart rate variability, respiratory rate, and body temperature were measured every 3 minutes for 15 minutes during manual restraint in 13 adult laying hens (Gallus gallus domesticus). Heart rate variability was significantly higher in hooded hens than in nonhooded hens (P= 0.003) but was not significant over time. Hooded hens were also found to have significantly lower heart rate (P = 0.043) and respiratory rate (P = 0.042) compared to nonhooded hens. Heart rate and respiratory rate significantly decreased over time, independent of the use of the hood (P = 0.008; P = 0.01, respectively). Temperature was found to increase significantly (P = 0.001) over time for both groups. Overall, hooding increased heart rate variability, a factor associated with a lower stress level, and decreased heart rate and respiratory rate. In conclusion, these data suggest that the use of the hood reduces stress levels in birds during manual restraint. Therefore, the use of the hood is encouraged for short (less than 15 minutes) painless procedures, such as physical examination or radiographic acquisition.