The aim of this study was to determine and compare the acute effects of exercise order during upper-lower body alternated supersets. This study was conducted by using quantitative time series experimental design. Twenty resistance-trained men performed different exercises order of upper body (bench press) and lower body (squat) exercises; (i) upper body to lower body (order A) and (ii) lower body to upper body (order B) in random arrangement for three sets with 120 s rest inter-set. All participants performed both exercises at 75% of their one repetition-maximum (1RM) value. Muscles activation and repetitions completed were recorded during both exercises order. Repeated measure analysis of variance (ANOVA) was used to analyse the different of all variables. Results showed order A produced higher upper body muscles activation (pectoralis major: ρ < .05, triceps brachii: ρ < .05) and number of repetitions completed (ρ < .05) in bench press for all three sets compared to order B. In contrast, order B showed higher lower body muscles activation (rectus femoris: ρ < .05, biceps femoris: ρ < .05) in squat compared to order A. Number of repetitions completed during squat were higher during order B compared to order A in the first set, ρ < .05. In conclusion, the results of this study suggested that the order of exercises performed in a resistance training session will determine the benefits gained. The findings of this study could be used as guideline for individuals involved in strength and conditioning to plan a better resistance training program for achieving their own specific goals.