In recent decades reported findings regarding gender differences in reading achievement, cognitive abilities and maturation process in boys and girls are conflicting. As reading is one of the most important processes in the maturation of an individual, the aim of the study was to better understand gender differences between primary school students. The study evaluates differences in Heart Rate Variability (HRV), Electroencephalography (EEG), Electrodermal Activities (EDA) and eye movement of participants during the reading task. Taking into account that colour may affect reading skills, in that it affects the emotional and physiological state of the body, the research attempts to provide a better understanding of gender differences in reading through examining the effect of colour, as applied to reading content. The physiological responses of 50 children (25 boys and 25 girls) to 12 different background and overlay colours of reading content were measured and summarised during the reading process. Our findings show that boys have shorter reading duration scores and a longer Saccade Count, Saccade Duration Total, and Saccade Duration Average when reading on a coloured background, especially purple, which could be caused by their motivation and by the type of reading task. Also, the boys had higher values for the Delta band and the Whole Range of EEG measurements in comparison to the girls when reading on coloured backgrounds, which could reflect the faster maturation of the girls. Regarding EDA measurements we did not find systematic differences between groups either on white or on coloured/overlay background. We found the most significant differences arose in the HRV parameters, namely (SDNN (ms), STD HR (beats/min), RMSSD (ms), NN50 (beats), pNN50 (%), CVRR) when children read the text on coloured/overlay backgrounds, where the girls showed systematically higher values on HRV measurements in comparison to the boys, mostly with yellow, red, and orange overlay colours.