Background Bra straps are a primary source of discomfort during sport and exercise, particularly for women with large breasts. This study aimed to investigate the effects of altering bra strap orientation and design on bra strap comfort, pressure and breast support in women with large breasts. This is a descriptive laboratory study. Methods Bra strap discomfort (visual analogue scale, 0 to 10), pressure (custom-designed 10 mm2 calibrated pressure sensor, 0.5 to 24 kPa range, 50 Hz, S2011, Novel GmbH, Munich, Germany, placed under the right bra strap at the crest of each participant's shoulder), preference ranking and vertical breast displacement (VBD; Optotrak Certus® motion capture system, 200 Hz, Northern Digital, Ontario, Canada) data during dynamic treadmill running and static upright standing (pressure only) were collected for 23 active women with large breasts (D+ cup size) while they wore an encapsulation sports bra with six different bra strap conditions (two bra strap orientations: vertical and cross-back; three bra strap designs: standard width, wide and gel). Results Bra strap discomfort was significantly less (p ≤ 0.001) in the vertical compared to the cross-back strap orientation, which was the most preferred orientation despite no significant difference in strap pressure. The wide strap design had the lowest discomfort scores, significantly lower strap pressure compared to the standard width and gel strap designs (p < 0.001), and was equally the most preferred design with the gel straps. There was no significant difference in VBD among the six strap conditions. Conclusions Bra straps that are vertically orientated and wide (approximately 4.5 cm in width) are preferable for women with large breasts during sport and exercise to minimise bra strap pressure and discomfort. The addition of gel pads under bra straps may also decrease discomfort and prevent straps slipping off the shoulders, although this notion warrants further investigation.