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The conceptual evolution of nicotinamide adenine dinucleotide (NAD⁺) from being seen as a simple metabolic cofactor to a pivotal cosubstrate for proteins regulating metabolism and longevity, including the sirtuin family of protein deacylases, has led to a new wave of scientific interest in NAD⁺. NAD⁺ levels decline during ageing, and alterations in NAD⁺ homeostasis can be found in virtually all age-related diseases, including neurodegeneration, diabetes and cancer. In preclinical settings, various strategies to increase NAD⁺ levels have shown beneficial effects, thus starting a competitive race to discover marketable NAD⁺ boosters to improve healthspan and lifespan. Here, we review the basics of NAD⁺ biochemistry and metabolism, and its roles in health and disease, and we discuss current challenges and the future translational potential of NAD⁺ research.