While tissue acidosis causes local deep-tissue pain, its effect on referred pain and mechanical muscle hyperalgesia is unknown. The aim of this study was to investigate a human experimental acidic muscle pain model using a randomized, controlled, single-blinded study design. Seventy-two subjects (36 female) participated in three visits, each involving one 15 min intramuscular infusion into the anterior tibialis muscle: acidic phosphate buffer (pH 5.2) at 40 ml/h (N=69) or 20 ml/h (N=54), normal phosphate buffer (pH 7.3) at 40 ml/h (N=70), or isotonic saline at 40 ml/h (N=19). Pain ratings and pressure sensitivity of superficial and deep tissues were assessed before, during, and 20 min after infusion. Acidic buffer produced light to moderate, rate-dependent, muscle pain (not sex-dependent) compared to the control infusions, that referred pain to the ankle in 80% of women and 40% of men. Pain did not vary across self-reported menstrual phases. Pressure pain thresholds (PPTs) were reduced over the infused muscle with acidic infusion, defined as primary mechanical hyperalgesia. PPTs decreased at the ankle in those with referred pain in response to acidic buffer, i.e. referred mechanical hyperalgesia, but not at the foot. No pain or changes in PPTs occurred in the contralateral leg. These results demonstrate muscle acidosis can lead to local and referred pain and hyperalgesia, with significant sex differences in development of referred pain.