In chronic musculoskeletal pain conditions, the balance between supraspinal facilitation and inhibition of pain shifts towards an overall decrease in inhibition. Application of a tonic painful stimulus results in activation of diffuse noxious inhibitory controls (DNIC). The aims of the present experimental human study were (1) to compare DNIC, evoked separately, by hypertonic saline (6%)-induced muscle pain (tibialis anterior) or cold pressor pain; (2) to investigate DNIC evoked by concomitant experimental muscle pain and cold pressor pain, and (3) to analyze for gender differences. Ten males and 10 age matched females participated in two sessions. In the first session unilateral muscle pain or unilateral cold pressor pain were induced separately; in the second session unilateral muscle pain and unilateral cold pressor pain were induced concomitantly. Pressure pain thresholds (PPT) were measured around the knee joint before, during, and after DNIC induction. Cold pressor pain increased PPT in both males and females with greater increases in males. Hypertonic saline-evoked muscle pain significantly increased PPT in males but not in females. When cold pressor and muscle pain were applied concomitantly the PPT increases were smaller when compared to the individual sessions. This study showed for the first time that two concurrent conditioning tonic pain stimuli (muscle pain and cold pressor pain) cause less DNIC compared with either of the conditioning stimuli given alone; and males showed greater DNIC than females. This may explain why patients with chronic musculoskeletal pain have impaired DNIC.