Exposure to additional environmental stress during computer work, such as visual and psychological demands, is associated with increased eye and neck discomfort, altered moods, and reduced well-being. The aim of this study is to elucidate further how subjective responses in healthy, young females with normal binocular vision are affected by glare and psychological stress during computer work, and to investigate possible associations between trapezius muscle blood flow and neck pain development. Methods Forty-three females participated in a laboratory experiment with a within-subject design. Four ten-minute computer work conditions with exposure to different stressors were performed at an ergonomically optimal workstation, under the following series of conditions: no additional stress, visual stress (induced as direct glare from a large glare source), psychological stress, and combined visual and psychological stress. Before and immediately after each computer work condition, questionnaires regarding different visual and eye symptoms, neck and shoulder symptoms, positive and negative state moods, perceived task difficulty, and perceived ambient lighting were completed. Associations between neck pain and trapezius muscle blood flow were also investigated. Results Exposure to direct glare induced greater development of visual/eye symptoms and discomfort, while psychological stress exposure made participants feel more negative and stressed. The perception of work lighting during glare exposure was closely related to perceived stress, and associations between visual discomfort and eyestrain, and neck pain were observed in all conditions. Furthermore, participants with high trapezius muscle blood flow overall reported more neck pain, independent of exposure. Conclusions Exposure to visual and psychological stresses during computer work affects the development of symptoms and negative moods in healthy, young females with normal binocular vision, but in different ways. The results also demonstrate the complex interactions involved in symptom development and lighting appraisal during computer work. When optimizing computer workstations, the complexity of the field must be taken into account, and several factors, including visual conditions, must be considered carefully.