Western diets rich in animal protein and poor in fruit and vegetables increase the body acid load, a predictor of type 2 diabetes risk. The relationships between dietary acid load, body acid/base balance and insulin resistance remain unclear. The objective of this study was to evaluate the association between dietary acid load, body acid/base balance and insulin resistance at baseline and following a short-term overfeeding in healthy individuals. Insulin sensitivity was measured by euglycaemic-hyperinsulinaemic clamp in healthy individuals (n=104, 49 men). Plasma lactate, an indicator of metabolic acidosis, was measured and acid load scores (potential renal acid load [PRAL] and net endogenous acid production [NEAP]) were derived from diet diaries. The cohort was grouped into lean and overweight/obese and the latter was further classified as insulin-sensitive (Obsen) and insulin-resistant (Obres) based on glucose infusion rate (GIR, top tertile vs. bottom 2 tertiles) during the clamp. A subset of 40 individuals underwent a 28-day overfeeding intervention (+1250 kcal/day) and studies were repeated. Obsen and Obres were matched for adiposity (BMI and fat mass, both P=1). Fasting plasma lactate was higher in Obres (0.78 [0.63-1.14] mmol/L) compared with both lean (0.71 [0.44-0.90] mmol/L, P=0.02) and Obsen (0.67 [0.56-0.79] mmol/L, P=0.04), and not different between lean and Obsen (P=0.9). Overfeeding was characterised by an increase in dietary acid load scores PRAL (P=0.003) and NEAP (P=0.05), a reduction in GIR (P=0.03) and an increase in fasting plasma lactate (P=0.02). The change in lactate was inversely associated with the change in GIR (r=-0.36, P=0.03). In conclusion, mild metabolic acidosis, measured by plasma lactate, aligns with insulin resistance independent of obesity and is induced by short-term increases in energy and dietary acid load in healthy humans. Further studies are required to determine whether buffering mild metabolic acidosis improves insulin resistance and reduces diabetes risk.