To dissociate the possible differential effects of negative energy balance and reduction in body fat mass (FM) on inflammatory markers: C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6), haptoglobin, transferrin and the adipokines leptin and adiponectin. Thirty-three obese subjects (BMI: 34.0+/-3.1 kg/m(2), age: 43.0+/-10.5 years, mean+/-s.d., 16 men) participated in a 20-week controlled dietary intervention divided into four periods. Weight reduction was induced by an 8-week low energy diet (3.4 MJ d(-1)) (LED-1) followed by a 4-week weight maintenance program (M-1). Subsequently participants underwent an additional 4-week LED (4.2 MJ d(-1)) (LED-2) followed by a final 4-week weight maintenance diet (M-2). Blood samples and anthropometrics were assessed at baseline and after LED-1, M-1, LED-2 and M-2. Body weight was significantly reduced by 13% (13.7+/-4.0 kg, P<0.0001) after LED-1. However, a reduction in high-sensitive CRP (hs-CRP) by 35% (-1.1 (95% CI: -2.5:0.2) mg l(-1), P=0.02) only became apparent after LED-2, which produced an additional weight loss of 2.9 kg compared to baseline, and it was maintained after M-2 (-1.0 (-1.4:0.4) mg l(-1), P=0.02). Also IL-6 was reduced by 21% (-0.6 (-2.4:0.2) ng l(-1), P=0.02) after M-2. The reductions in hs-CRP and IL-6 were both associated with reduction in FM but not body weight. Haptoglobin, transferrin and leptin were significantly reduced after both LED-1 and LED-2, but increased during weight maintenance. Adiponectin was not significantly changed during the intervention. The results suggest that, whereas haptoglobin and transferrin respond more rapidly and are more susceptible to the acute change in energy balance, a reduction in hs-CRP and IL-6 seems to be achieved by a reduction in FM when a new steady state has been established.