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OBJECTIVE:Area of muscle, fat, and bone is often measured in thigh CT scans when tissue composition is a key outcome. SliceOmatic software is commonly referenced for such analysis but published methods may be insufficient for new users. Thus, a quick start guide to calculating thigh composition using SliceOmatic has been developed. METHODS:CT images of the thigh were collected from older (69 ± 4 yrs, N = 24) adults before and after 12-weeks of resistance training. SliceOmatic was used to segment images into seven density regions encompassing fat, muscle, and bone from -190 to +2000 Hounsfield Units [HU]. The relative contributions to thigh area and the effects of tissue density overlap for skin and marrow with muscle and fat were determined. RESULTS:The largest contributors to the thigh were normal fat (-190 to -30 HU, 29.1 ± 7.4%) and muscle (35 to 100 HU, 48.9 ± 8.2%) while the smallest were high density (101 to 150 HU, 0.79 ± 0.50%) and very high density muscle (151 to 200 HU, 0.07 ± 0.02%). Training significantly (P<0.05) increased area for muscle in the very low (-29 to -1 HU, 5.5 ± 7.9%), low (0 to 34 HU, 9.6 ± 16.8%), normal (35 to 100 HU, 4.2 ± 7.9%), and high (100 to 150 HU, 70.9 ± 80.6%) density ranges for muscle. Normal fat, very high density muscle and bone did not change (P>0.05). Contributions to area were altered by ~1% or less and the results of training were not affected by accounting for skin and marrow. CONCLUSIONS:When using SliceOmatic to calculate thigh composition, accounting for skin and marrow may not be necessary. We recommend defining muscle as -29 to +200 HU but that smaller ranges (e.g. low density muscle, 0 to 34 HU) can easily be examined for relationships with the health condition and intervention of interest. TRIAL REGISTRATION:Clinicaltrials.gov NCT02261961.