This pilot study investigated the impact of a 24-h sleep deprivation period on circulating biomarkers of creatine metabolism in 23 healthy adults (24.6 ± 4.3 years; 7 females). Contrary to the hypothesis that sleep deprivation would reduce creatine levels, serum creatine significantly increased post-deprivation (95% CI −3.66 to 25.22 μmol/L; P = 0.03), while guanidinoacetic acid and creatinine levels remained unchanged. Our findings suggest a complex relationship between sleep deprivation and creatine metabolism, emphasizing the need for further research using advanced biomarkers to elucidate these mechanisms and assess creatine’s potential protective role under stress conditions.