Prospective studies have indicated an age-related impairment of the immune response. Carotenoids have been hypothesised to enhance immune cell function. The aim of the present study was to describe the age-related effects and the impact of in vivo dietary carotenoid depletion and repletion on specific and non-specific immunity. A total of ninety-eight healthy male subjects (aged 20–75 years) received a carotenoid-depleted diet for 3 weeks and were then supplemented daily for 5 weeks with 30 mg β-carotene, 15 mg lycopene and 9 mg lutein. Blood samples were collected at study entry, after depletion and supplementation, and biomarkers of immune status were determined. We found that serum IgA levels were positively correlated with ageing. Lymphocyte phenotyping indicated an increase with age in the memory T-helper cell subpopulation (CD4+CD45RO+) concomitantly with a decrease in naive T-helper cells (CD4+CD45RA+). A significant increase in the natural killer cells subpopulation and a small decrease in B lymphocytes were also observed, especially for the oldest volunteers. From ex vivo cell function exploration, a positive correlation was observed between age and IL-2 production of phytohaemagglutinin-stimulated lymphocytes. Neutrophils' bactericidal activity was significantly impaired with age (from 50 years) and was modulated by carotenoid status. An age effect was found on neutrophils' spontaneous migration but not on directed migration. Immune response in healthy human subjects is mostly affected by age rather than by dietary carotenoid depletion and repletion. Even in carefully selected healthy volunteers, some age-related immune changes occur predominantly from 50 years onwards. This immunosenescence could generate a loss in the immune system adjustment capacity.