Background: It is questionable how repeated patch tests with nickel sulfate in infancy affect nickel patch test reactivity at a later age. Methods: The Danish Allergy Research Center (DARC) cohort encompasses 562 infants invited to a clinical examination including patch tests with nickel sulfate six times during the first 36 months of life. At the follow-up investigation at 14 years of age (2013–2014), participants were offered re-patch tests with nickel sulfate. The Odense Adolescence Cohort Study TOACS cohort encompasses 1501 schoolchildren evaluated for the first time at 14 years of age (1995–1996) including clinical examination and nickel sulfate patch tests. The prevalence of nickel sensitization in the DARC cohort was compared to the prevalence in the TOACS cohort at 14 years of age. Results: Nickel sulfate sensitization was found in 1.2% of the participants from the DARC cohort tested repeatedly with nickel sulfate in early childhood and retested at 14 years of age compared to 8.6% of the participants from the TOACS cohort patch-tested for the first time at 14 years of age using the same patch test system and test concentration. Conclusion: The significant difference in nickel patch test reactivity comparing the two cohorts may reflect an immunologic effect or the effect of nickel regulation.