Newly hatched chicks may be held longer than 48 h and experience long periods of fasting in commercial hatcheries. Limited information is known about the physiological status of chicks in such situations, due to the difficulty of precisely recording time of hatch. This study investigated the effect of the time from hatch to pulling (holding period) on physiological measures/parameters in 109 broiler chicks. Fertile Ross 308 eggs were incubated in a custom built small-scale incubator. The individual hatching time of each focal chick was determined using eggshell temperature monitoring. At ‘pulling’ (512 h of incubation time), the quality of focal chicks was assessed using the chick scoring method and physiological parameters were measured including BW, organ (heart, liver and stomach) weights, blood values and plasma corticosterone level. The time from hatch to pulling varied from 7.58 to 44.97 h. Egg weight at setting was significantly correlated with chick BW and weight of organs at pulling, but had no effect on chick quality, blood values and plasma corticosterone. Relative BW at pulling was negatively associated with the duration of holding period (P=0.002). However, there was a positive correlation between relative stomach weight and the duration of the holding period (P<0.001). As the holding period duration increased, there was a trend that blood partial pressure of oxygen, haematocrit and haemoglobin also increased, and blood partial pressure of carbon dioxide, total carbon dioxide and bicarbonate decreased (P<0.05). A wide range of plasma corticosterone was observed from chicks that had experienced different durations of holding period. We conclude that shortening the hatch window and minimising the number of chicks that experience a long holding period before pulling may improve chick quality and physiological status, which may be due to unfavourable environmental conditions that include feed and water deprivation.