With the recent growth in the development of augmented reality (AR) technologies , it is becoming important to study human perception of AR scenes. In order to detect whether users will suffer more from visual and operator fatigue when watching virtual objects through optical see-through head-mounted displays (OST-HMDs), compared with watching real objects in the real world, we propose a comparative experiment including a virtual magic cube task and a real magic cube task. The scores of the subjective questionnaires (SQ) and the values of the critical flicker frequency (CFF) were obtained from 18 participants. In our study, we use several electrooculogram (EOG) and heart rate variability (HRV) measures as objective indicators of visual and operator fatigue. Statistical analyses were performed to deal with the subjective and objective indicators in the two tasks. Our results suggest that participants were very likely to suffer more from visual and operator fatigue when watching virtual objects presented by the OST-HMD. In addition, the present study provides hints that HRV and EOG measures could be used to explore how visual and operator fatigue are induced by AR content. Finally, three novel HRV measures are proposed to be used as potential indicators of operator fatigue.