Heel lifts are a treatment option for low back pain (LBP), whilst high-heeled shoes have been linked to LBP development. This study evaluated the effects of in-shoe 20 mm high bilateral heel lifts on trunk muscle activity. Activity of the erector spinae (ErSp), internal oblique and external oblique muscles was evaluated using surface electromyography in 15 young (20.7+/-0.9 years) healthy female participants. Measures were taken during overground gait, both immediately and following two days habituation to the heel lifts. Immediately following the addition of the heel lifts, levels of ErSp muscle activity in the 5% epoch following heel strike increased by 19.2% (p<0.05). Following habituation, levels of ErSp muscle activity in the 5% epoch prior to heel strike increased by 24.1% (p<0.05), and a 14 ms (p<0.001) earlier onset of ErSp muscle activity prior to heel strike was observed. These results indicate the heel lifts altered muscle activity reactively around heel strike (i.e. greater activity after heel strike) immediately after application and proactively (i.e. earlier onsets and greater activity prior to heel strike) after short term habituation. When put in context of previous research on trunk muscle activity in LBP populations, these changes may be important considerations for the aetiology, treatment and prevention of LBP.