Exaggerated fear-reactions are associated with injurious flying, smothering, feather pecking and other events that compromise animal welfare in laying hens. The aim of this study was to test the hypothesis that chicks with access to litter during the first five weeks of life would be less fearful as adult hens compared to birds reared without access to litter. The hypothesis was tested in a national on-farm study in commercial aviary flocks in Norway. Five rearing farmers divided the pullets into two groups within their rearing houses. While the chicks were enclosed inside the aviary rows during the first five weeks of life, paper substrate where food and other particles could accumulate, covered the wire mesh floor in the treatment group, whereas the control group was reared on bare wire mesh. At 30 weeks of age, 23 aviary flocks (11 control flocks reared without paper and 12 treatment flocks reared with paper) were visited. During the visit, the fearfulness of the adult birds was tested in a stationary person test and a novel object test. The data was analysed by ANOVA or logistic regression as appropriate. The access to litter during rearing did not influence the number of birds that approached within 25 cm of the stationary person (p = 0.51). All flocks, regardless of rearing treatment, had birds which came within 2 m of the stationary person. The latency to approach within 2 m of the stationary person tended to be influenced by provision of environmental enrichment as adults (p = 0.08) and by the interaction between treatment × rearing farm (p = 0.08). The number of birds that approached within 2 m of the stationary person was influenced by the interaction between treatment during rearing and provision of enrichment as adults (p = 0.03), however, the post hoc test showed no pairwise differences. All flocks, regardless of rearing treatment, had birds that approached the novel object. The access to litter during rearing did not influence the birds’ latency to approach the novel object. The number of birds approaching the novel object was affected by the interaction between access to substrate during rearing and provision of environmental enrichment as adults (p = 0.05). The results indicate that both adding paper substrate to chicks from the first day of life and environmental enrichment as adults, reduce fearfulness in laying hens.