Background: Treadmill animal models are commonly used to study effects of exercise on bone. Since mechanical loading induces bone strain, resulting in bone formation, exercise that induces higher strains is likely to cause more bone formation. Our aim was to investigate the effect of slope and additional load on limb bone strain. Methods: Horizontal and vertical ground reaction forces on left fore-limb (FL) and hind-limb (HL) of twenty 23-week old female Wistar rats (weight 279 ± 26 g) were measured for six combinations of SLOPE (−10°, 0°, +10°) and LOAD (0 to 23% of body mass). Peak force (Fmax), rate of force rise (RC), stance time (Tstance) and impulse (Fint) on FLs and HLs were analyzed. Results: For the FL, peak ground reaction forces and rate of force rise were highest when walking downward −10° with load (Fmax = 2.09±0.05 N, FLRC = 34±2 N/s) For the HL, ground reaction forces and rate of force rise were highest when walking upward +10°, without load (Fmax = 2.20±0.05 N, HLRC = 34±1 N/s). Load increased stance time. Without additional load, estimates for the highest FL loading (slope is −10°) were larger than for the highest HL loading (slope is +10°) relative to level walking. Conclusions: Thus, walking downward has a higher impact on FL bones, while walking upward is a more optimal HL exercise. Additional load may have a small effect on FL loading.