With COVID-19, evidence indicates that the elderly will have worse biochemical markers related to health in social isolation. The objective was to analyze the impacts on physical fitness and biochemical parameters of older adults’ health during COVID-19 social isolation. Quantitative, longitudinal, and observational study was conducted between 2020, 2021, and 2022. Thirty-three older adults of both sexes were evaluated. A sociodemographic questionnaire, biomarkers, and health-related physical fitness were used. Significant differences were observed for the sum of maximum isometric right and left handgrip strength, with a reduction in 2022 (p = 0.009); getting up and walking (p < 0.001), reduction in 2021 and 2022 (p < 0.05); elbow flexion and extension (p = 0.004), reduction in 2021 (p = 0.006); and sitting and standing (p = 0.002), reduction in 2022 (p = 0.003) and peak oxygen consumption (p < 0.001), reduction in 2021 and 2022 (p < 0.05). Differences were observed in fasting blood glucose (p < 0.001), with increase in 2021 and 2022 (p < 0.05), triglycerides (p < 0.001), with increase in 2021 and 2022 (p < 0.05), triglyceride–glucose index (p < 0.001), with increase in 2021 and 2022 (p < 0.05), triglyceride–glucose index with waist circumference (p < 0.001), with increase in 2021 (p < 0.001); and triglyceride–glucose index with body mass index (p < 0.001), with increase in 2021 (p < 0.001). However, no differences were observed between anthropometric and body composition (p > 0.05). Conclusions: Older people had changes in biochemical and physical fitness parameters related to health during the social isolation of the COVID-19 pandemic.