Contact with green space in the environment has been associated with mental health benefits, but the mechanism underpinning this association is not clear. This study extends an earlier exploratory study showing that more green space in deprived urban neighbourhoods in Scotland is linked to lower levels of perceived stress and improved physiological stress as measured by diurnal patterns of cortisol secretion. Salivary cortisol concentrations were measured at 3, 6 and 9 h post awakening over two consecutive weekdays, together with measures of perceived stress. Participants (n = 106) were men and women not in work aged between 35–55 years, resident in socially disadvantaged districts from the same Scottish, UK, urban context as the earlier study. Results from linear regression analyses showed a significant and negative relationship between higher green space levels and stress levels, indicating living in areas with a higher percentage of green space is associated with lower stress, confirming the earlier study findings. This study further extends the findings by showing significant gender differences in stress patterns by levels of green space, with women in lower green space areas showing higher levels of stress. A significant interaction effect between gender and percentage green space on mean cortisol concentrations showed a positive effect of higher green space in relation to cortisol measures in women, but not in men. Higher levels of neighbourhood green space were associated with healthier mean cortisol levels in women whilst also attenuating higher cortisol levels in men. We conclude that higher levels of green space in residential neighbourhoods, for this deprived urban population of middle-aged men and women not in work, are linked with lower perceived stress and a steeper (healthier) diurnal cortisol decline. However, overall patterns and levels of cortisol secretion in men and women were differentially related to neighbourhood green space and warrant further investigation.