Rationale: Older adults are potentially engaging in greater amounts of prolonged sedentary behavior (SB) during the COVID-19 pandemic. Given a poor tolerance for intense exercise, SB displacement with light intensity physical activity (LIPA) is a promising health enhancing alternative. Therefore, the aims of this study were to investigate the effects of two different types of SB displacement on health outcomes in older adults and any differential impact of associated LIPA pattern. Method: 28 older women (age: 73 ± 5 years, BMI: 26.1 ± 3.6 kg/m2) underwent overnight fasted dual energy x-ray absorptiometry (DEXA) imaging, blood sampling, and functional assessments before being randomly allocated to one of two groups: (1) single continuous bout of 45–50 min LIPA daily (n = 14); or (2) SB fragmentation (SBF; ~48 min LIPA daily, 2 min LIPA for every 30 min of SB; n = 14). Compliance was systematically monitored using tri-axial accelerometery. All measures were taken at weeks 0 and 8. Results: Physical behavior significantly altered (decreased SB/increased LIPA; p < 0.05) and to a similar extent in both groups. We observed a significant reduction in serum triglycerides [p = 0.045, effect size (ɳp2) = 0.15; SBF: −0.26 ± 0.77 mmol/L, LIPA: −0.26 ± 0.51 mmol/L], improved 30 s sit-to-stand (STS) count (p = 0.002, ɳp2 = 0.32, 2 ± 3 STS) and speed (p = 0.009, ɳp2 = 0.35, −10 ± 33%), as well as increased average handgrip strength (p = 0.001, ɳp2 = 0.45, 6 ± 12%), and gait speed (p = 0.005, ɳp2 = 0.27, 0.09 ± 0.16 m/s) in both groups. Interestingly, SBF caused a greater increase in peak handgrip strength (8 ± 14%), compared to LIPA (2 ± 10%; p = 0.04, ɳp2 = 0.38). Conclusion: SB displacement induced significant improvements in fasting triglycerides, gait speed, as-well as STS endurance/speed in older women. Frequent vs. continuous SB displacement also caused greater increases in handgrip strength. While both SB displacement protocols display promise as efficacious home-based interventions for self-isolating older adults, our results would suggest a physical functioning advantage of the SBF protocol for certain outcomes.