All color-difference formulas are developed to evaluate color differences for pairs of stimuli with hairline separation. In printing applications, however, color differences are frequently judged between a pair of samples with no separation (NS) because they are printed adjacently on the same piece of paper. A new formula, ΔENS, has been developed for pairs of stimuli with NS. An experiment was conducted to investigate the effect of different color-difference magnitudes using sample pairs with NS. 1012 printed pairs with NS were prepared around 11 CIE recommended color centers. The pairs, representing four color-difference magnitudes of 1, 2, 4, and 8 CIELAB units were visually evaluated by a panel of 19 observers using the gray-scale method. Comparison of the present data based on pairs with NS, and previously generated data using pairs with hairline separation, showed a clear separation effect. A new color-difference equation for the NS viewing condition (ΔENS) is proposed by modifying the CIEDE2000 formula. The separation effect can be well described by the new formula. For a sample pair with NS, when the CIEDE2000 color difference is less than 9.1, a larger color difference leads to a larger lightness difference, but has no effect on the chromatic difference. When the CIEDE2000 color difference is greater than 9.1, the effect is the opposite. The new formula is recommended for future research to evaluate its performance in appropriate applications.