Background: The Centers for Disease Control and Prevention has designated physical inactivity as an actual cause of chronic disease, although there is little direct physiological evidence proving that reductions in daily physical activity increase the risk of disease. Therefore we wanted to determine the metabolic consequences of a reduced number of daily steps. Design: Ten healthy young men decreased their daily activity level from a median of 10,053 (range 6,854 -13,218) to 1,381 (range 1,061 -1,658) steps/day for 2 weeks. Pre- and post-test were done during ambulatory care at the research institution. The period with reduced stepping was implemented in the participant’s daily living. Dietary records were taken throughout to ensure that habitual dietary intake was maintained. The study was performed according to the Declaration of Helsinki and approved by The Scientific-Ethical Committee of Copenhagen and Frederiksberg Municipalities, and participants provided written informed consent. Method: The study’s primary outcomes were 1) insulin sensitivity measured with a hyperinsulinemic-euglycemic clamp with stable isotopes, 2) insulin signalling (tyrosine (tyr) phosphorylation (p) of the insulin receptor (IR) beta (pTyrIRbeta), pThr308Akt, and pAS160) in skeletal muscle biopsies (Western blotting), and 3) maximal oxygen consumption (VO2max) and body composition (measured by DXA scanning). Statistical analysis was done by analysis of variance followed by Bonferroni-corrected t tests or by paired t-tests. Results: A reduced number of daily steps induced a decline in peripheral insulin sensitivity (Paired t-test p < 0.01) without an effect on endogenous glucose production. Insulin-stimulated pThr308Akt decreased after pace reduction (ANOVA p<0.01) with a post hoc analysis revealing the most pronounced effect after 4 h of insulin infusion (p<0.05). There was no change in pTyrIRbeta and pAS160 and the protein expression of IR, Akt, and AS160 was not affected by insulin stimulation or intervention. In addition, a 7% decline in VO2max (ml/min) (Paired t-test p < 0.01) and a decline in fat-free mass (1.2 kg) (Paired t-test p < 0.001) were found. Conclusion: One biological cause for the enormous public health problem of type 2 diabetes and central obesity has been identified. A reduction in daily physical activity as implemented by a reduction in the number of daily steps in healthy young men for only two weeks induces insulin resistance with a reduction in insulin-stimulated pAktthr308; this effect may represent one link between an inactive lifestyle and increased risk of chronic disease.