It is unclear how white adipose tissue (WAT) inflammatory signaling proteins respond during the early stages of overnutrition.To investigate the effect of short-term, high-fat overfeeding on fasting abdominal subcutaneous WAT total content and phosphorylation of proteins involved in nuclear factor-κB (NF-κB) inflammatory signaling, systemic metabolic measures and inflammatory biomarkers.Individuals consumed a high-fat (65% total energy total fat), high-energy (50% above estimated energy requirements) diet for 7 days.Fifteen participants (age 27 ± 1 y; BMI 24.4 ± 0.6 kg/m2) completed the study. Body mass increased following high-fat overfeeding (+1.2 ± 0.2 kg; P < 0.0001). However, total content and phosphorylation of proteins involved in NF-κB inflammatory signaling were unchanged following the intervention. Fasting serum glucose (+0.2 ± 0.0 mmol/L), total cholesterol (+0.4 ± 0.1 mmol/L), low-density lipoprotein cholesterol (+0.3 ± 0.1 mmol/L), high-density lipoprotein cholesterol (+0.2 ± 0.0 mmol/L), and lipopolysaccharide-binding protein (LBP; +4.7 ± 2.1 µg/mL) increased, whereas triacylglycerol concentrations (-0.2 ± 0.1 mmol/L) decreased following overfeeding (all P < 0.05). Systemic biomarkers (insulin, soluble cluster of differentiation 14 (CD14), C-reactive protein, IL-6, TNF-α and monocyte chemoattractant protein-1) and the proportion and concentration of circulating CD14+ monocytes were unaffected by overfeeding.Acute lipid oversupply did not impact on total content or phosphorylation of proteins involved in WAT NF-κB inflammatory signaling, despite modest weight gain and metabolic alterations. Systemic LBP, which is implicated in the progression of low-grade inflammation during the development of obesity, increased in response to a 7-day high-fat overfeeding period.