PurposePhysical exercise improves physical and mental health; however, the differences between voluntary and forced exercise protocols are unclear. In addition, knowledge regarding the consequences of differences in testing timing, such as light and dark phases, in response to exercise type is limited. We investigated the effects of chronic forced and voluntary wheel running on the changes in brain monoamine levels (5-HT: serotonin, DA: dopamine, NA: noradrenaline), anxiety-like behaviours, and physiological stress responses in the light and dark phases. MethodsAdult male Wistar rats were equally and randomly assigned to four groups: sedentary control, voluntary exercise (free running on a wheel, V-EX), voluntary limited exercise (wheel available only 1h/day, VL-EX), and forced exercise (running on a motorised wheel, F-EX). Each group was further divided into dark- or light-experimental condition groups. After 4 weeks, the rats underwent an open-field test. The monoamines and their metabolite levels were measured in the major neural cell bodies and the projection areas related to behaviour, cognition, anxiety, and stress in the brain. ResultsAdrenal hypertrophy and elevated body temperature, except during the exercise period, were observed in the F-EX rats that exhibited anxiety-like behaviour. The levels of monoamines and their metabolites, particularly the 5-HTergic and DAergic systems, in specific areas, were significantly altered in the rats in the V-EX group compared to those in the VL-EX and other groups. These differences were observed only in the dark phase. ConclusionThe results suggest that V-EX mainly stimulates the 5-HTergic and DAergic systems, while F-EX induces physiological stress and increases anxiety-like behaviour during the dark phase. This study highlights the importance of accounting for exercise types and light/dark phases in behavioural neuroscience experiments.