Previous findings of decreased mental and perceptual motor performance during parabolic flights have been attributed mainly to the primary effects of weightlessness rather than the accompanying effects of stress and altered mood. Although recent studies have alluded to the possible negative effects of stress on performance, there has been no attempt to investigate this during parabolic flights. Over a period of 3 years, 27 human participants (male n = 18, mean age +/- SD 34.67 +/- 7.59 years; female n = 9, 36.22 +/- 9.92 years) were recruited with the aim to evaluate if, and to what extent, parabolic flights are accompanied by changes in mood. Furthermore, the relationships between mood and physiological markers of stress and arousal, namely circulating stress hormones (ACTH, cortisol, epinephrine, norepinephrine, prolactin and brain activity (EEG)) were investigated. A strong and significant correlation was found between circulating stress hormone concentrations and perceived physical state, motivational state (MOT) and psychological strain (PSYCHO), whereas no interaction between mood and EEG or EEG and stress hormone concentrations was observed. Therefore, two different stress responses appear to be present during parabolic flight. The first seems to be characterised by general cortical arousal, whereas the second seems to evolve from the adrenomedullary system. It is likely that both these mechanisms have different effects on mental and perceptual motor performance, which require further investigation and should to be taken into account when interpreting previous weightlessness research.