Muscle hyperalgesia was induced by combining low-intensity muscle insult with 6 min of fatiguing contractions as previously described [21 (link)]. On day 0, mice were anesthetized with 2–4% isoflurane and an intramuscular (i.m.) injection of 20μl normal saline adjusted to pH 5.0 or 7.2 (control group) were given. On day 5, mice were anesthetized with 2–4% isoflurane, needles electrodes were placed in the gastrocnemius muscle belly and 6 min of submaximal fatiguing contractions were given through a Grass stimulator (7V intensity, 40 Hz for 3.75s, 4.25 s between contractions; Grass Technologies, WesWarwick, RI). Immediately after the end of fatiguing muscle contractions, a second i.m. injection of 20 μl pH 5.0 or 7.2 normal saline were given. The hindpaw was attached to a force plate connected to a force transducer to quantify force output (iWorx, Dover, NH). Fatigue was quantified by examining the % decline in maximum force to three 100 Hz stimuli at 7 V by comparing forces before and after the fatiguing stimuli. We previously show that unbuffered pH 5.0 saline injections reduce muscle pH to approximately 6.9 [64 (link)] and neither two injections of pH 5.0 alone nor fatigue alone produce hyperalgesia [21 (link),76 (link)].