BACKGROUND: In the wild, mice are subject to changes in light intensity and spectrum (colour) across the solar day. In addition, mice are able to self-modulate their light exposure - a concept termed light sampling behaviour, which results in intermittent patterns of light exposure. These complexities are poorly considered in most laboratory animal housing. As such, our understanding of the role of intermittent exposure to naturally-occurring changes in intensity and spectrum in circadian behaviour are limited. To address these issues we simulated both daylight and twilight in the laboratory, and provided a dark nestbox to enable behavioural regulation of light exposure. RESULTS: The results show that gradual changes in light intensity are a key driver of crepuscular light sampling in mice, whilst demonstrating for the first time that spectral cues at twilight modulate the timing of behaviour - advancing locomotor activity by 0.5h and light sampling behaviour by 1.1h. CONCLUSIONS: Collectively, our results demonstrate the significance of changes in intensity and spectrum across twilight for regulating mouse behaviour. These findings highlight important differences in mouse behaviour under naturalistic environments compared to normal laboratory conditions.