The present study was conducted to investigate the impact of a 42d period of heat stress on laying hens with respect to rectal temperature, body-surface temperature, heart rate, heterophil to lymphocyte ratio (H/L ratio), corticosterone (CORT) in plasma and yolk samples, and video-assessed behaviors. A total of one hundred twenty 56-week-old ISA-brown laying hens were continuously exposed to one of three temperature regimes: optimal (LT; 22°C), moderate (MT; 27°C), and high (HT; 32°C). The relative humidity was maintained at 50% in all treatments. HT vs. MT and LT increased rectal temperature, heart rate, and body-surface temperature on all days (P<0.05). HT vs. LT raised (P<0.05) the H/L ratio in blood at day 42 following heat exposure. On the other hand, both HT- and MT-exposed laying hens had higher (P<0.05) plasma CORT compared with those on LT group only at 28 and 42d following heat treatment. Yolk CORT was elevated (P<0.05) in the HT vs. MT and LT groups at 3 and 14d following heat treatment. Panting and wing elevation were the most relevant behaviors in laying hens exposed to HT vs. MT and LT. The study shows that rectal and body-surface temperature, heart rate, and behavior, but not CORT or H/L ratio, are the reliable indicators for assessing the stress status of laying hens over extended period of heat stress.