Objective: The aim of this study was to determine the intra-rater and inter-rater reliability of the dual rehabilitative ultrasound imaging (D-RUSI) when simultaneously measuring muscle thickness changes at rest and during co-contraction of the biceps brachii (BB) and triceps brachii (TB). Design: Cross-sectional study. Methods: This study included 36 healthy participants (23 men, 13 women). The participants sat on a chair in a comfortable position with a cushion placed under their elbow to maintain a 90-degree elbow flexion angle. The muscle thickness of the biceps brachii and triceps brachii was measured twice using the D-RUSI by two examiners during resting and co-contraction states. One week later, the same procedure was performed once again. Results: The intra-class correlation coefficients (ICCs) for intra-rater reliability ranged from 0.887 to 0.989 and the confidence interval was within an acceptable range of 0.778 to 0.994. The standard error of measurement (SEM) values ranged from 0.303 to 0.866 and the minimal detectable change (MDC) values ranged from 0.84 to 2.40. The ICCs for inter-rater reliability ranged from 0.758 to 0.925. The SEM values ranged from 0.702 to 1.486 and the MDC values ranged from 1.95 to 4.12. Conclusions: The use of the D-RUSI of the BB muscle had a very good intra-rater reliability and very good inter-rater reliability at the resting state, and a, good inter-rater reliability at the co-activation state. ICC values showed very good intra-reliability and inter-reliability for the TB muscle. the D-RUSI is a useful tool for simultaneously measuring the thickness of two muscles when the BB is an agonist and the TB is an antagonist during co-activation of the upper arm.