Purpose: Most studies on barefoot and shod walking have so far focused on leg muscle activity. However, footwear might also have an impact on the back and neck. The aim of the present study was to compare back and neck muscle activity as well as kinematic gait parameters during barefoot walking, conventional shod walking and walking in flexible shoes, commercially designed with the intention to imitate barefoot walking. Methods: Thirty healthy subjects (16 male and 14 female, mean age 31.4 AE 12.8 years) walked at self-selected speed over an instrumented walkway mat, either barefoot, with their own casual shoes or with these flexible shoes. Muscle activity was measured by EMG from the lumbar iliocostalis muscle, the lumbar longissimus muscle, the lumbar multifidi muscles, the trapezius muscle (pars descendens), the neck extensor muscles and the sternocleidomastoideus muscle. Results: The results indicated that back and neck muscle activity was slightly lower when wearing conventional footwear as opposed to walking barefoot or in the flexible shoe. Both the conventional and the flexible shoe, in contrast, significantly altered the kinematic gait parameters. Conclusions: Back and neck muscle activity was slightly influenced by footwear. The relevance of these findings should be investigated in the long term by longitudinal studies with healthy subjects as well as with patients suffering from lower back and neck pain.