The prevention of type 2 diabetes in persons at risk for diabetes is of utmost importance. Physical activity in general and even exercises at moderate intensities such as walking significantly reduce the risk of the development of type 2 diabetes. However, it is still a matter of debate whether lipids and glucose metabolism are differently affected by regular concentric (e.g., uphill walking) and eccentric (e.g., downhill walking) endurance exercise. The aim of this study was to investigate the effects of short-term (3 weeks) uphill and downhill walking on glucose metabolism and blood lipids in pre-diabetic middle-aged men in a real world setting. The study was designed as an investigator-initiated 2 group random selection pre-test post-test trial. Sixteen pre-diabetic men (age: 56.9 ± 5.1 years; BMI: 28.1 ± 2.3 kg·m^-2) performed 9 uphill (n = 8) or 9 downhill (n = 8) walking sessions within 3 weeks. The primary outcomes were the markers of glucose metabolism and blood lipids measured before and after the training period. After uphill walking glucose tolerance (area under the curve of the oral glucose tolerance test: -43.25 ± 53.12 mg·dl^-1; p = 0.05; effect size: 0.81), triglycerides (-48.75 ± 54.49 mg·dl^-1; p = 0.036; effect size: 0.89), HDL-C (+7.86 ± 9.54 mg·dl^-1; p = 0.05; effect size: 0.82) and total cholesterol/HDL-C ratio (-0.58 ± 0.41; p = 0.012; effect size: 1.39) had significantly improved. No significant metabolic adaptations were found after downhill walking. However, when adjusted for estimated energy expenditure, uphill and downhill walking had equal effects on almost all metabolic parameters. Moreover, the magnitude of the baseline impairments of glucose tolerance was significantly related to the extent of change in both groups. Depending on the fitness level and individual preferences both types of exercise may be useful for the prevention of type 2 diabetes and disorders in lipid metabolism.