This study was conducted to compare subjective and objective assessment methods of a two-colored chewing gum test and to find out whether these methods are capable of discriminating masticatory performances between sexes. 31 adults, 16 males and 15 females participated in this study. Each subject chewed five samples of two-colored chewing gum sticks for 5, 10, 20, 30 and 50 chewing strokes, respectively. The subjective color-mixing and shape indices for the gum bolus (SCMI-B, SSI-B) and the subjective color-mixing index and objective color-mixing ratio for the gum wafer (SCMI-W, OCMR-W) were evaluated by two independent examiners and, on a different day, re-evaluated by one of the examiners. The SCMI-B and SCMI-W assessments had inter- and intra-examiner reliable agreement at 20 or more chewing strokes. The OCMR-W measurement demonstrated high accuracy and low reproducibility between and within the examiners. There were significant gender differences in the distribution of SCMI-W scores (P = 0.044) and in the mean OCMI-W (P = 0.007). The SCMI-B and SCMI-W assessments and the OCMR-W measurement were reliable and valid at the 20 and 30 chewing strokes in this two-colored chewing gum test. The subjective color-mixing index (SCMI-W) and objective color-mixing ratio (OCMR-W) for the chewing gum wafer are capable of discriminating masticatory performance between sexes in this two-colored chewing gum test and that the OCMR-W measurement is discriminating better than the SCMI-W assessment.