Nicotinamide adenine dinucleotide (NAD+) is a cofactor with roles in many biochemical processes. NAD+ can be synthesised from exogenous dietary sources and via endogenous salvage pathways. Regulators of this system include nicotinamide phosphoribosyltransferase (NAMPT), nicotinamide riboside kinase 2 (NMRK2), nicotinamide n-methyltransferase (NNMT), and nicotinamide mononucleotide adenylyltransferase 1/3 (NMNAT1/3). Ageing is marked by a decline in NAD+ biosynthesis, contributing to metabolic dysfunction. Thus, developing strategies to regulate NAD+ biosynthesis across the lifespan has garnered interest. Sprint interval training (SIT) has emerged as a potent metabolic stimulus, inducing beneficial musculoskeletal adaptations. Low energy conditions, such as the fasted state, have been investigated for their potential to upregulate NAD+ biosynthetic pathway activity. This study compared the effects of fasted (FAST) and carbohydrate-fed (FED) SIT on NAD+ biosynthetic pathway gene expression in skeletal muscle of recreationally active males. This study received local ethical approval and was conducted in line with the Declaration of Helsinki. Healthy, recreationally active (VO2 max < 50 ml.kg-1.min-1) males (n=18) were randomised to complete SIT under FAST or FED conditions. Participants attended the lab after an overnight fast (≥10 h) and ingested 0.33 g.kg-1 body mass (BM) of non-caloric placebo (FAST), or 0.91 g.kg-1 BM maltodextrin (FED). Forty-five minutes post feeding, participants underwent SIT consisting of 4 X 30 s “all out” cycle sprints at a resistance of 7.5 % BM, interspersed with 4 min recovery. Muscle biopsies were obtained under local anaesthetic from m. vastus lateralis at rest (pre-feeding) and 3 h post-exercise. RNA extracted from skeletal muscle underwent multiplex PCR analysis to determine mRNA expression of NAMPT, NMRK2, NNMT, NMNAT1, NMNAT3, and the reference gene UBE2D2. Paired and independent t-tests determined within and between-group differences in gene expression fold changes from baseline. NMRK2, NAMPT, and NNMT expression increased in both groups following SIT (p < 0.05). NMNAT1 was unchanged and NMNAT3 was decreased from baseline in FAST only (p < 0.05). No between-groups differences were observed for ΔNMRK2, ΔNMNAT1, or ΔNMNAT3. Both ΔNAMPT and ΔNNMT were upregulated to a greater extent in FAST compared with FED (p < 0.05). In summary, these findings indicate that SIT is a potent regulator of a network of genes modulating NAD+ biosynthesis and that there is divergent regulation of some, but not all these genes in response to FAST compared with FED SIT. This may have implications for health across the lifespan. Future research should investigate the efficacy of FAST compared with FED SIT on the regulation of this system in ageing populations.