Skeletal muscle is a highly dynamic tissue of critical importance for general health. Indeed, skeletal muscles play key roles in posture, mobility, thermogenesis and whole-body glucose homeostasis. As the largest amino acid reservoir of the human body, skeletal muscles also play essential roles during catabolic conditions. Therefore, developing strategies and interventions to improve or maintain skeletal muscle health is of paramount importance. Nicotinamide riboside supplementation (NRS) is currently one of the nutritional supplementations attracting the most attention in this regard. NR is a naturally occurring vitamin B3 and a precursor of NAD+. Energy metabolism in skeletal muscle is highly regulated by NAD+ and its reduced form NADH. Indeed, many dehydrogenases of key metabolic pathways, including beta-oxidation, glycolysis and the Kreb's cycle, require NAD+ for their catalytic activities. Apart from their roles in cell metabolism, NAD+ and NADH also regulate multiple processes and pathways, including DNA repair, sirtuin activity as well as mitochondrial biogenesis and function (Fang et al. 2017). The strong interest that currently surrounds NRS is based on preclinical studies conducted in Caenorhabditis elegans, Drosophila and rodents that overall have shown that NR supplementation can improve mitochondrial health, prolong lifespan and ameliorate the pathophysiology of several ageing-related diseases such as Alzheimer's and Parkinson's disease [see Fang et al. (2017) for a detailed reviews]. NRS was also shown in mice to enhance oxidative metabolism and protect against high-fat diet-induced obesity (Fang et al. 2017). Based on these discoveries, increasing NAD+ levels has emerged as a promising strategy to protect mitochondrial function, improve lifespan and ameliorate the outcome of many diseases as well as metabolic complications associated with obesity, such as insulin resistance. Further strengthening the interest that surrounds NRS, Martens et al. (2018) recently showed that NRS is well tolerated and effectively increases NAD+ concentration in circulating peripheral blood mononuclear cells, confirming the ability of NRS to increase NAD+ bioavailability in humans. However, whether NRS can improve insulin resistance and ameliorate mitochondrial health in humans remains largely unexplored. In a recent randomized control trial (RCT) which led to three publications, one in the present issue of the Journal of Physiology (Dollerup et al. 2020), one in The Journal of Clinical Endocrinology and Metabolism (Dollerup et al. 2019) and one in the American Journal of Clinical Nutrition (Dollerup et al. 2018), Dollerup and collaborators investigated whether 12 weeks of NRS (2000 mg/day) could improve whole-body glucose metabolism as well as skeletal muscle mitochondrial respiration and content in obese and insulin-resistant men. In their papers, Dollerup and colleagues report data indicating that although NRS increases in urinary metabolites of NR (Dollerup et al. 2018), it failed to increase NAD+ content in skeletal muscles (Dollerup et al. 2020). Using the permeabilized fibre approach, the current gold standard approach to assess mitochondrial respiration in situ, they report that NRS failed to enhance mitochondrial respiration (Dollerup et al. 2020). They also report that the content of robust and well-validated markers of mitochondrial content were unaffected by NRS (Dollerup et al. 2020). Similarly, using confocal laser scanning microscopy, they also report that mitochondrial morphology was unaffected by NRS in their participants (Dollerup et al. 2020). Accordingly, no impact of NRS on the content of protein-regulating mitochondrial dynamics were observed (Dollerup et al. 2020). In addition to the absence of an impact of NRS on mitochondrial respiration, content and morphology/dynamics, Dollerup and colleagues also report that the content of key proteins involved in insulin-sensitivity (such as GLUT4, hexokinase II, mTOR and glycogen synthase) were unaffected by NRS (Dollerup et al. 2020). In line with these findings, NRS failed to improve insulin sensitivity and glucose tolerance, as assessed using the hyperinsulinaemic–euglycaemic clamp and an oral glucose tolerance test (OGTT), respectively (Dollerup et al. 2018, 2019). In addition, no effects of NRS were observed on circulating levels of pancreatic hormones (i.e. insulin, C-peptide and glucagon) or incretin hormones (i.e. glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide) during the OGTT (Dollerup et al. 2019), indicating that NRS did not improve pancreatic secretory function and incretin hormone secretion. Taken together, the findings collected by Dollerup and collaborators provide robust evidence that 12 weeks of NRS does not improve skeletal muscle mitochondrial respiration and whole-body glucose homeostasis in a population of obese and insulin-resistant men. In support of this conclusion, research recently published in Cell Reports shows in healthy elderly individuals that 250 mg of NRS twice a day for 21 days does not improve skeletal muscle mitochondrial bioenergetics and whole-body glucose homeostasis (Elhassan et al. 2019). Collectively, these findings therefore challenge preclinical data obtained in animal models and indicate that NRS is ineffective in enhancing mitochondrial respiration and whole-body glucose homeostasis in obese insulin-resistant men. Although further studies are warranted to definitively conclude on the potential health benefit of NAD+ precursors, the RCT from Dollerup and colleagues indicate that health professionals and the general population should await further scientific evidence before recommending or engaging in NRS. Further studies will also be required to define whether tissue-specificity in the NRS effect exists in humans and whether NRS can exert beneficial effects in other populations with differences in age, sex or pre-existing diseases. No competing interests declared. All authors were involved in the conception or design of the work and drafting or revising it critically for important intellectual content. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. GG is supported by a Chercheur Boursier Junior 1 salary award from the Fonds de Recherche du Québec en Santé (FRQS-35184).