Animal and human experimental studies have suggested the importance of spatial summation in the nociception processing and in the activation of descending inhibition. However, the relationship between the areas (size) of muscles stimulated and the recruitment of descending inhibition has not been addressed. Consequently, we tested whether bilateral versus unilateral injection of hypertonic saline into trapezius muscles caused hypoalgesia to pressure pain (pressure pain thresholds, PPTs) in the local pain areas (the trapezius muscles) and the referred pain areas (the posterolateral neck muscles). Two groups of volunteers participated. One group received a unilateral injection (one injection) and the other group bilateral injections (two injections). In the bilateral group, hypertonic saline was injected in one trapezius first, and 45 s later, while pain was still present from the first injection, a second injection was performed into the contralateral trapezius muscle. The saline-evoked time to maximal pain was significantly shorter after the second injection than after the first injection. More subjects developed referred pain after the bilateral compared with the unilateral injection. In the referred pain areas, the PPTs 7.5 and 15 min after the second injection were significantly increased compared with the first injection, while no changes in the PPT were observed in local and referred pain areas after unilateral injection. This suggests that the induction of descending inhibition was triggered by spatial summation during the later phase of experimentally induced muscle pain. The present experimental model might be used for further investigation of descending inhibition related to the spatial characteristics of nociceptive stimuli in humans.