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Human-robot collaboration (HRC) is an emerging research area that has gained tremendous attention from both academia and industry. Since some robot-related factors can elicit mental stress or have negative psychological effects on human workers, it is essential to understand these factors and maintain workers’ mental stress at a low level. Galvanic Skin Response (GSR) measures skin conductance and is known to be a physiological measurement that reflects short-term mental stress. Typically, skin conductance increases in response to greater mental stress. In this study, the mental stress caused by the hand-over activities of a collaborative robot was investigated using both GSR as an objective measurement and NASA-Task Load Index (TLX) as a subjective assessment. Several robot-related factors that may lead to mental stress were experimentally examined. GSR outcomes indicated that end effector approaching within workers’ view, low end effector speed, and constrained end effector trajectory led to a significantly lower skin conductance. Some aspects of the NASA-TLX also indicated that speed and trajectory significantly affected the scores. Yet, no significant differences were found between approaching directions regarding NASA-TLX scores.