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The lack of a pathophysiological marker hinders studies on environmental illnesses of unknown origin. Hence, research focused on the identification of such a marker is a priority. This study investigated the nature and a possible etiology of fatigue in hypersensitivity to electricity (the most commonly reported environmental illness in Sweden). The aim was to test the hypothesis that perceived fatigue was due to alterations in cholinesterase activity. The study group consisted of 14 people who reported a hypersensitivity to electricity, including disabling fatigue. We assessed cholinesterase activity three times: twice based on current symptoms reported by the subjects (severe fatigue attributed to electromagnetic fields and absence of this symptom) and once at a randomly selected time. No significant reduction in acetylcholinesterase was identified in any subject. Examined on a group level, no significant reduction in activity was identified at the time of severe fatigue, and no correlation between reported degree of fatigue and cholinesterase activity was observed. Fatigue attributed to electromagnetic fields was nonphysical and showed a significant correlation to difficulties in concentrating. The results do not support the hypothesis that a change in cholinesterase activity mediates fatigue in people reporting hypersensitivity to electricity.