Since the beginning of the 20th century there has been a scientific debate about the potential effects of air ions on biological tissues, wellbeing and health. Effects on the cardiovascular and respiratory system as well as on mental health have been described. In recent years, there has been a renewed interest in this topic. In an experimental indoor setting we conducted a double-blind cross-over trial to determine if higher levels of air ions, generated by a special wall paint, affect cognitive performance, wellbeing, lung function, and cardiovascular function. Twenty healthy non-smoking volunteers (10 female, 10 male) participated in the study. Levels of air ions, volatile organic compounds and indoor climate factors were determined by standardized measurement procedures. Air ions affected the autonomous nervous system (in terms of an increase of sympathetic activity accompanied by a small decrease of vagal efferent activity): In the test room with higher levels of air ions (2194/cm3 vs. 1038/cm3) a significantly higher low to high frequency ratio of the electrocardiography (ECG) beat-to-beat interval spectrogram was found. Furthermore, six of nine subtests of a cognitive performance test were solved better, three of them statistically significant (verbal factor, reasoning, and perceptual speed), in the room with higher ion concentration. There was no influence of air ions on lung function and on wellbeing. Our results indicate slightly activating and cognitive performance enhancing effects of a short-term exposure to higher indoor air ion concentrations.