Background <p>Multi-frequency bioelectrical impedance analysis (MFBIA) methods offer reliable and moderately accurate estimates of body composition in tightly controlled conditions (prandial and hydration status, recent exercise, time of day).</p> Objective <p>This study examined MFBIA reliability and validity in a real-world environment where these factors were not controlled.</p> Methods <p>Regional and total body composition estimates by MFBIA (InBody 770) were compared to dual-energy X-ray absorptiometry (DXA) in 1000 healthy adults (667 men; 333 women), including fat mass (FM), percent body fat (%BF), fat-free mass (FFM), and visceral adipose tissue (VAT). In subsets, reliability was determined from duplicate MFBIA and DXA obtained within 1 week, and total body water (TBW) was compared to single-frequency BIA (SFBIA).</p> Results <p>MFBIA demonstrated modest population-level agreement with DXA for total body FM (men, <i>r</i> = 0.93, bias −3.7 ± 2.6 kg; women, <i>r</i> = 0.96, bias, −1.9 ± 1.8 kg), %BF (men, <i>r</i> = 0.89, bias, −4.2 ± 3.0%; women, <i>r</i> = 0.92, bias, −2.8 ± 2.6%), and FFM (men, <i>r</i> = 0.95, bias, 3.4 ± 2.8 kg; women, <i>r</i> = 0.94, bias, 2.0 ± 2.2 kg). Regional correlations were highest for trunk FM (men, <i>r</i> = 0.92, CCC = 0.86; women <i>r</i> = 0.93, CCC = 0.93) and lowest for VAT (men, <i>r</i> = 0.74, CCC = 0.68; women, <i>r</i> = 0.74, CCC = 0.34). DXA and MFBIA regional and total assessments were highly reliable (DXA, ICC 0.990–0.998) and (MFBIA, ICC 0.987–0.995). TBW by MFBIA and SFBIA showed moderate agreement (men, <i>r</i> = 0.73, bias, −1.89 ± 3.31; women, <i>r</i> = 0.82, bias, −1.74 ± 2.01).</p> Conclusion <p>This MFBIA system was shown to have high retest reliability and, when compared to laboratory methods, provides a moderately accurate method for measuring TBW and body composition (except for VAT) in uncontrolled conditions.</p>