Egg storage longer than 7 days is associated with negative effects on hatchability traits. Pre-storage incubation has been a suggested method to reduce the negative effects of long-term storage times by enhancing the developmental stage of the embryo and probably reducing the embryonic stress. The objective of the present study was to investigate the effects of pre-storage incubation and storage time on hatchability characteristics, chick quality and serum thyroid hormones, antioxidative properties and immunoglobulin Y (IgY) concentrations of newly hatched chicks at two breeder flock ages. A total of 8000 fertile eggs were obtained from two different ages of chicken breeder hens (Egyptian local cross, Inshas). Half of the eggs were collected from young breeder hens (28 weeks old) and the other half from old breeder hens (50 weeks old). In each breeder flock age, eggs were distributed in a completely randomized experimental design in a 2×4 factorial arrangement, with two storage periods (4 or 14 days) and four pre-storage incubation durations (0, 4, 6 or 8 h at 37.5°C). At 28 and 50 weeks of age, pre-storage incubation and its interaction with storage period influenced significantly the apparent fertility, hatchability of set eggs and hatchability of fertile eggs and this improvement in hatchability is attributed to the reduction in embryonic mortality (early, intermediate and late). Pre-storage incubation for 6 or 8 h elevated significantly the grade A chicks and reduced the grade B chicks in comparison with non-heated controls. Interestingly, for eggs stored for 14 days, pre-storage incubation for 6 or 8 h enhanced serum triiodothyronine, thyroxine, glutathione peroxidase activity, total antioxidant capacity and IgY concentrations significantly and decreased serum malondialdehyde concentration significantly in the newly hatched chicks. It could be concluded that pre-storage incubation enhanced the hatching results, improved the antioxidative properties, reduced lipid peroxidation and elevated the humoral immunity in the newly hatched chicks. Hence, several benefits might be gained by pre-storage incubation when fertilized eggs will be stored for long periods.