Background <p>Pathogenic variants in <i>RYR1</i> cause a spectrum of rare congenital myopathies associated with intracellular calcium dysregulation. Glutathione redox imbalance has been reported in several <i>Ryr1</i> disease model systems and clinical studies. NAD⁺ and NADP are essential cofactors in cellular metabolism and redox homeostasis. NAD⁺ deficiency has been associated with skeletal muscle bioenergetic deficits in mitochondrial myopathy and sarcopenia.</p> Methods <p>Using a new colorimetric assay and large control dataset (<i>n</i> = 299), we assessed redox balance (glutathione, NAD⁺, and NADP) in whole blood from 28 <i>RYR1</i>-RM affected individuals (NCT02362425). Analyses were expanded to human skeletal muscle (<i>n</i> = 4), primary myotube cultures (<i>n</i> = 5), and whole blood and skeletal muscle specimens from <i>Ryr1</i> Y524S mice. The in vitro effects of nicotinamide riboside (NR) on cellular NAD⁺ content and mitochondrial respirometry were also tested.</p> Results <p>At baseline, a majority of affected individuals exhibited systemic NAD⁺ deficiency (19/28 [68%] &lt; 21&#xa0;µM) and increased NADPH concentrations (22/26 [85%] &gt; 1.6&#xa0;µM). When compared to controls, decreased NAD⁺/NADH and NADP/NADPH ratios were observed in 9/28 and 23/26 individuals, respectively. In patient-derived myotube cultures (<i>n</i> = 5), NR appeared to increase cellular NAD⁺ concentrations in a dose and time-dependent manner at 72-h only and favorably modified maximal respiration and ATP production. Average whole blood GSH/GSSG ratio was comparable between groups, and redox imbalance was not observed in <i>Ryr1</i> Y524S specimens.</p> Conclusions <p>NAD⁺ and NADP dyshomeostasis was identified in a subset of <i>RYR1</i>-RM affected individuals. Further experiments are warranted to confirm if NAD⁺ repletion could be an attractive therapeutic approach given the favorable outcomes reported in other neuromuscular disorders.</p> Graphical Abstract <p></p>