Numerous academics and research findings have described the purpose and benefits of hill training. However, the current body of knowledge is insufficient to confidently inform the formulation of suggestions that explain which hills repeat, long-gradual, intermediate or short-steeps provide the most benefits. Therefore, the aim of this study was to investigate the effects of eight-week uphill training: shallow hill training (~ 2.5%), intermediate hill training (~ 5.1%), and steeper hill training (~ 7.6%) on bio-motor skills (maximal velocity, running performance, and strength endurance performance), physiological (maximal oxygen consumption, the velocity at maximal oxygen consumption, resting heart rate, and recovery heart rate), and biomechanical (step rate, step length, contact time, and flight time) of middle-distance runners. A pre-post parallel true experimental research design was employed. Forty moderately trained middle-distance runners were selected using a cencus sampling technique and randomly assigned to the three intervention groups, or control groups (n = 10 each). Over 8 weeks, participants completed gradient-specific training and outcomes were assessed. The result in two-way mixed RM-ANOVA revealed a significant main effect of time and hill gradients on bio-motor skills, physiological and biomechanical measures of middle-distance runners. The findings showed that uphill training has a significant positive effect on bio-motor skills, physiological and biomechanical measures of running performance, while the steeper uphill shows the most significant benefit compared to the lower hill gradients. Therefore, coaches and athletes should customize uphill training programs while carefully matching the strengths and weaknesses of the athlete and the demands of the event.