This study investigated whether variations in gradient would affect the magnitude of physiological responses during a 30-minute run at an intensity of 70% of maximal oxygen capacity ( O 2max ). Forty untrained collegiate men were randomly assigned into 0%, −5%, −11% and −16% groups ( n = 10 per group), and then performed a 30-minute run at gradients of 0%, −5%, −11%and −16%, respectively, at the intensity of 70%of their predetermined O 2max . Oxygen consumption ( O 2 ), minute ventilation ( E ), respiratory exchange ratio (RER), heart rate (HR), and rating of perceived exertion (RPE) were measured at 5, 10, 15, 20, 25 and 30 minutes, respectively, during each run. Blood lactate (LA) concentration was assessed by fingertip blood sample at 3 minutes after each run. The results showed that elevations in O 2 , E , RER and HR during running for the −11% and −16% groups were greater ( p < 0.05) than for the −5% and 0% groups. For the −11% group, elevations of these measures were greater ( p < 0.05) than those of these measures for the −5% group. However, the changes in these measures showed no significant difference ( p > 0.05) between the 0% and −5% groups, or between the −11% and −16% groups. As for RPE and LA, no significant differences ( p > 0.05) among the groups were observed. It is concluded that the steeper the gradient, the greater the increases in O 2 , E , RER and HR. This may be due to the fact that at a steeper downhill gradient (−16%), the quadriceps femoris muscle lengthens to a greater extent than at lower (−5%, −11%) and level gradients.