The current study investigated the affects of acute alterations in extracellular osmolality on resting in-vitro skeletal muscle. Isolated, whole muscle strips (Soleus and EDL) from 5 week old Long Evans rats were incubated for 60 min in an organ bath containing Sigma Medium-199 (resting tension (1g), bubbled with 95:5 O2:CO2, 30 ± 2°C, and pH 7.4). Three different osmotic states were used; an iso-osmotic (290±10 Osm) (CON), hyper-osmotic (400 ±10 Osm) (HYPR), and hypo-osmotic (190 ± 10 Osm) (HYPO). After incubation, the relative water content decreased with HYPR, and increased with HYPO in both muscle types. As a result of the HYPO type I fiber area was increased in the Soleus (HYPO = 4066.3 ± 634.4μm2, CON = 2990 ± 364.1 μm2, n=12, p<0.05), whereas only TYPE II fiber area was increased in the EDL (HYPO = 2419.3 ± 681.5 μm2, CON = 1729.1 ± 513.1μm2, n>11, p<0.001). HYPR resulted in increased muscle lactate concentrations (EDL, p<0.001; Soleus, p<0.001, n=51) in both muscle types, but no extracellular lactate content was detected. Glycogen content decreased in EDL as a result of HYPR (HYPR=79.3 ± 11.8 mmol/kg, ISO=94.3.3 ± 16.1, p<0.01), whereas glycogen content in the soleus increased in HYPO (HYPO= 92.5 ± 12.2 mmol/kg, ISO=76.6 ± 18.5 mmol/kg, p<0.05). ATP and PCr concentrations confirmed muscle viability. Therefore, acute exposure of skeletal muscle to extracellular osmolality increases intracellular concentrations of lactate. The specific mechanisms underlying these changes in lactate remain to be determined. Supported by NSERC, Canada.