Introduction: Chronic and acute short sleep associate with many adverse effects. Metabolic and neuroendocrine effects during the day following short sleep, however, are unknown. Methods: Randomized cross-over study using short sleep (4h) vs normal sleep (8h) in 31 males (31 years (IQR 23-47, BMI 26.0±4.2 kg/m2 (range 20.1-35.0). Glucose, lipid, incretin, ACTH, and cortisol levels, as well as respiratory exchange ratio (RER; indirect calorimetry) were assessed following standardized mixed meals. RNAseq of morning skeletal muscle biopsies and pathway analysis on differentially expressed genes was performed. Results: Short sleep increased fasting free fatty acids (0.34 mmol/L (IQR 0.26 - 0.38) vs 0.47 mmol/L (IQR 0.38 - 0.70), P<0.0001). Cortisol levels peaked upon early wake-up, which eliminated a peak at normal wake-up. Morning postprandial glucose peaked later (time P<0.001, levels P=0.106, pattern P=0.031), whilst insulin was unaffected. Afternoon postprandial insulin was lowered (time P<0.001, levels P=0.044, pattern P=0.419), whilst glucose was unaffected. Morning and afternoon postprandial TC, and LDL-c were lowered (morning: TC, time P<0.0001, levels P=0.009, pattern P=0.289; LDL-c, time P<0.001, levels P=0.038, pattern P=0.797; afternoon: TC, time P<0.001, levels P=0.007, pattern P=0.92; LDL-c, time P<0.001, levels P=0.055, pattern P=0.991). Pathway analysis of muscle revealed increased fatty acid oxidation (FAO), reflected by increased Oxidative Phosphorylation and Mitochondrial Dysfunction pathways, accompanied by lowered fasting RER (0.87 (IQR 0.86 - 0.92) vs 0.86 (IQR 0.84 - 0.87), P=0.0033). Conclusions: Acute partial sleep restriction subtly and temporarily changed glucose dynamics (morning), and insulin levels (afternoon), and lowered TC and LDL-c levels in the morning and afternoon, with concurrent increased FAO and mitochondrial dysfunction pathways in skeletal muscle, and calorimetric shifting from glucose towards lipid oxidation (fasted and postprandial), potentially ...